This document provides an overview of ultra performance liquid chromatography (UPLC). UPLC uses sub-2 μm particles and operates at higher pressures than HPLC to dramatically increase resolution, sensitivity, and speed of analysis while maintaining practicality. Key advantages of UPLC include decreased run times, increased sensitivity and throughput, and reduced solvent consumption compared to HPLC. The document discusses UPLC instrumentation including pumps, injectors, columns, detectors, and applications such as amino acid analysis and analysis of natural products and herbal medicines.
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.
fluid chromatography (SFC) can be used on an analytical
scale.
It is a combination of High performance liquid chromatography (HPLC)
and Gas chromatography (GC).
It can be used with non-volatile and thermally labile analytes.
It can be used with the universal flame ionization detector.
It is important to producing narrower peaks due to rapid diffusion.
It is important for the chiral separations and analysis of high-molecularweight
hydrocarbons.
Supercritical fluids are suitable as a substitute for organic solvents in a
range of industrial and laboratory processes.
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.
fluid chromatography (SFC) can be used on an analytical
scale.
It is a combination of High performance liquid chromatography (HPLC)
and Gas chromatography (GC).
It can be used with non-volatile and thermally labile analytes.
It can be used with the universal flame ionization detector.
It is important to producing narrower peaks due to rapid diffusion.
It is important for the chiral separations and analysis of high-molecularweight
hydrocarbons.
Supercritical fluids are suitable as a substitute for organic solvents in a
range of industrial and laboratory processes.
SFC ie. Supercritical Fluid Chromatography is one of the chromatographic technique. This presentation will help you to understand the basic principle behind it.
Reviews the SFC technique along with applications and case studies.
Use of animations disturbs the view. For full ppt, drop me a mail at varadbende96@gmail.com
a brief introduction to countercurrent chromatography with its principle. working and modes of operation along with little bit of history, the types of CCC and its applications
High Performance Liquid Chromatography (HPLC)VaibhaviPatil21
HPLC stands for High Performance Liquid Chromatography. previously HPLC was known as High pressure Liquid Chromatography as high pressure is required to pump the liquid,due to its efficiency ,later it came to know as High Performance liquid chromatography.It is an analytical technique which is used to analyze compunds in samples in pharma industry,chemical industry as well as in Bioanalytical Labs for studying BABE studies.
It is the form of Liquid column chromatography.In HPLC,the mobile phase is liquid and the stationary phase is solid.
HPLC can be divided into two types-
1) Normal phase HPLC
2) Reverse phase HPLC
HPLC can further be combined with Mass Spectrometry (LC-MS) which allows to seperate the samples with the analysis of mass.
HPLC PPT for you If You want to download it then download it .
Its is in original PPT form
Share it & improve your Knowledge.
INTRODUCTION
HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution.
HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector.
Compounds are separated by injecting a sample mixture into the column. The different component in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase.
The mobile phase must be degassed to eliminate the formation of air bubbles.
Counter current chromatography (CCC) is a liquid chromatography technique that
uses two immiscible liquid phases and no solid support.
2. One liquid acts as the stationary phase and the other as the mobile phase.
3. In Dual Flow CCC/CPC both liquid phases are flowing, as would be common in counter
current process extractors.
4. The liquid stationary phase(s) is held in place by gravity or by centrifugal force. The
gravity method is called droplet counter current chromatography (DCCC).
5. There are two modes of centrifugal force CCC: hydrostatic and hydrodynamic. In the
hydrostatic method.
6. The column is spun about
SFC ie. Supercritical Fluid Chromatography is one of the chromatographic technique. This presentation will help you to understand the basic principle behind it.
Reviews the SFC technique along with applications and case studies.
Use of animations disturbs the view. For full ppt, drop me a mail at varadbende96@gmail.com
a brief introduction to countercurrent chromatography with its principle. working and modes of operation along with little bit of history, the types of CCC and its applications
High Performance Liquid Chromatography (HPLC)VaibhaviPatil21
HPLC stands for High Performance Liquid Chromatography. previously HPLC was known as High pressure Liquid Chromatography as high pressure is required to pump the liquid,due to its efficiency ,later it came to know as High Performance liquid chromatography.It is an analytical technique which is used to analyze compunds in samples in pharma industry,chemical industry as well as in Bioanalytical Labs for studying BABE studies.
It is the form of Liquid column chromatography.In HPLC,the mobile phase is liquid and the stationary phase is solid.
HPLC can be divided into two types-
1) Normal phase HPLC
2) Reverse phase HPLC
HPLC can further be combined with Mass Spectrometry (LC-MS) which allows to seperate the samples with the analysis of mass.
HPLC PPT for you If You want to download it then download it .
Its is in original PPT form
Share it & improve your Knowledge.
INTRODUCTION
HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution.
HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector.
Compounds are separated by injecting a sample mixture into the column. The different component in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase.
The mobile phase must be degassed to eliminate the formation of air bubbles.
Counter current chromatography (CCC) is a liquid chromatography technique that
uses two immiscible liquid phases and no solid support.
2. One liquid acts as the stationary phase and the other as the mobile phase.
3. In Dual Flow CCC/CPC both liquid phases are flowing, as would be common in counter
current process extractors.
4. The liquid stationary phase(s) is held in place by gravity or by centrifugal force. The
gravity method is called droplet counter current chromatography (DCCC).
5. There are two modes of centrifugal force CCC: hydrostatic and hydrodynamic. In the
hydrostatic method.
6. The column is spun about
ICH Guidelines for Pharmacovigilance.pdfNEHA GUPTA
The "ICH Guidelines for Pharmacovigilance" PDF provides a comprehensive overview of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines related to pharmacovigilance. These guidelines aim to ensure that drugs are safe and effective for patients by monitoring and assessing adverse effects, ensuring proper reporting systems, and improving risk management practices. The document is essential for professionals in the pharmaceutical industry, regulatory authorities, and healthcare providers, offering detailed procedures and standards for pharmacovigilance activities to enhance drug safety and protect public health.
Global launch of the Healthy Ageing and Prevention Index 2nd wave – alongside...ILC- UK
The Healthy Ageing and Prevention Index is an online tool created by ILC that ranks countries on six metrics including, life span, health span, work span, income, environmental performance, and happiness. The Index helps us understand how well countries have adapted to longevity and inform decision makers on what must be done to maximise the economic benefits that comes with living well for longer.
Alongside the 77th World Health Assembly in Geneva on 28 May 2024, we launched the second version of our Index, allowing us to track progress and give new insights into what needs to be done to keep populations healthier for longer.
The speakers included:
Professor Orazio Schillaci, Minister of Health, Italy
Dr Hans Groth, Chairman of the Board, World Demographic & Ageing Forum
Professor Ilona Kickbusch, Founder and Chair, Global Health Centre, Geneva Graduate Institute and co-chair, World Health Summit Council
Dr Natasha Azzopardi Muscat, Director, Country Health Policies and Systems Division, World Health Organisation EURO
Dr Marta Lomazzi, Executive Manager, World Federation of Public Health Associations
Dr Shyam Bishen, Head, Centre for Health and Healthcare and Member of the Executive Committee, World Economic Forum
Dr Karin Tegmark Wisell, Director General, Public Health Agency of Sweden
How many patients does case series should have In comparison to case reports.pdfpubrica101
Pubrica’s team of researchers and writers create scientific and medical research articles, which may be important resources for authors and practitioners. Pubrica medical writers assist you in creating and revising the introduction by alerting the reader to gaps in the chosen study subject. Our professionals understand the order in which the hypothesis topic is followed by the broad subject, the issue, and the backdrop.
https://pubrica.com/academy/case-study-or-series/how-many-patients-does-case-series-should-have-in-comparison-to-case-reports/
Empowering ACOs: Leveraging Quality Management Tools for MIPS and BeyondHealth Catalyst
Join us as we delve into the crucial realm of quality reporting for MSSP (Medicare Shared Savings Program) Accountable Care Organizations (ACOs).
In this session, we will explore how a robust quality management solution can empower your organization to meet regulatory requirements and improve processes for MIPS reporting and internal quality programs. Learn how our MeasureAble application enables compliance and fosters continuous improvement.
India Clinical Trials Market: Industry Size and Growth Trends [2030] Analyzed...Kumar Satyam
According to TechSci Research report, "India Clinical Trials Market- By Region, Competition, Forecast & Opportunities, 2030F," the India Clinical Trials Market was valued at USD 2.05 billion in 2024 and is projected to grow at a compound annual growth rate (CAGR) of 8.64% through 2030. The market is driven by a variety of factors, making India an attractive destination for pharmaceutical companies and researchers. India's vast and diverse patient population, cost-effective operational environment, and a large pool of skilled medical professionals contribute significantly to the market's growth. Additionally, increasing government support in streamlining regulations and the growing prevalence of lifestyle diseases further propel the clinical trials market.
Growing Prevalence of Lifestyle Diseases
The rising incidence of lifestyle diseases such as diabetes, cardiovascular diseases, and cancer is a major trend driving the clinical trials market in India. These conditions necessitate the development and testing of new treatment methods, creating a robust demand for clinical trials. The increasing burden of these diseases highlights the need for innovative therapies and underscores the importance of India as a key player in global clinical research.
Telehealth Psychology Building Trust with Clients.pptxThe Harvest Clinic
Telehealth psychology is a digital approach that offers psychological services and mental health care to clients remotely, using technologies like video conferencing, phone calls, text messaging, and mobile apps for communication.
Defecation
Normal defecation begins with movement in the left colon, moving stool toward the anus. When stool reaches the rectum, the distention causes relaxation of the internal sphincter and an awareness of the need to defecate. At the time of defecation, the external sphincter relaxes, and abdominal muscles contract, increasing intrarectal pressure and forcing the stool out
The Valsalva maneuver exerts pressure to expel faeces through a voluntary contraction of the abdominal muscles while maintaining forced expiration against a closed airway. Patients with cardiovascular disease, glaucoma, increased intracranial pressure, or a new surgical wound are at greater risk for cardiac dysrhythmias and elevated blood pressure with the Valsalva maneuver and need to avoid straining to pass the stool.
Normal defecation is painless, resulting in passage of soft, formed stool
CONSTIPATION
Constipation is a symptom, not a disease. Improper diet, reduced fluid intake, lack of exercise, and certain medications can cause constipation. For example, patients receiving opiates for pain after surgery often require a stool softener or laxative to prevent constipation. The signs of constipation include infrequent bowel movements (less than every 3 days), difficulty passing stools, excessive straining, inability to defecate at will, and hard feaces
IMPACTION
Fecal impaction results from unrelieved constipation. It is a collection of hardened feces wedged in the rectum that a person cannot expel. In cases of severe impaction the mass extends up into the sigmoid colon.
DIARRHEA
Diarrhea is an increase in the number of stools and the passage of liquid, unformed feces. It is associated with disorders affecting digestion, absorption, and secretion in the GI tract. Intestinal contents pass through the small and large intestine too quickly to allow for the usual absorption of fluid and nutrients. Irritation within the colon results in increased mucus secretion. As a result, feces become watery, and the patient is unable to control the urge to defecate. Normally an anal bag is safe and effective in long-term treatment of patients with fecal incontinence at home, in hospice, or in the hospital. Fecal incontinence is expensive and a potentially dangerous condition in terms of contamination and risk of skin ulceration
HEMORRHOIDS
Hemorrhoids are dilated, engorged veins in the lining of the rectum. They are either external or internal.
FLATULENCE
As gas accumulates in the lumen of the intestines, the bowel wall stretches and distends (flatulence). It is a common cause of abdominal fullness, pain, and cramping. Normally intestinal gas escapes through the mouth (belching) or the anus (passing of flatus)
FECAL INCONTINENCE
Fecal incontinence is the inability to control passage of feces and gas from the anus. Incontinence harms a patient’s body image
PREPARATION AND GIVING OF LAXATIVESACCORDING TO POTTER AND PERRY,
An enema is the instillation of a solution into the rectum and sig
One of the most developed cities of India, the city of Chennai is the capital of Tamilnadu and many people from different parts of India come here to earn their bread and butter. Being a metropolitan, the city is filled with towering building and beaches but the sad part as with almost every Indian city
The dimensions of healthcare quality refer to various attributes or aspects that define the standard of healthcare services. These dimensions are used to evaluate, measure, and improve the quality of care provided to patients. A comprehensive understanding of these dimensions ensures that healthcare systems can address various aspects of patient care effectively and holistically. Dimensions of Healthcare Quality and Performance of care include the following; Appropriateness, Availability, Competence, Continuity, Effectiveness, Efficiency, Efficacy, Prevention, Respect and Care, Safety as well as Timeliness.
1. Patil V. P. et al. IRJP 2 (6) 2011 39-44
IRJP 2 (6) June 2011 Page 39-44
INTERNATIONAL RESEARCH JOURNAL OF PHARMACY ISSN 2230 – 8407
Available online http://www.irjponline.com
Review Article
ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY: A REVIEW
Patil V. P.1
*, Tathe R. D1
., Devdhe S. J.2
, Angadi S. S.2
and Kale S. H.3
1
Department of Pharmaceutical Analysis, Yash Institute of Pharmacy, Aurangabad, Maharashtra, India
2
Department of Pharmacology, Yash Institute of Pharmacy, Aurangabad, Maharashtra, India
3
Department of Pharmaceutical Chemistry, Yash Institute of Pharmacy, Aurangabad, Maharashtra, India
Article Received on: 09/04/2011 Revised on: 18/05/2011 Approved for publication: 10/06/2011
*Prof. Vandana P. Patil, Yash Institute of Pharmacy, Aurangabad-431134, Maharashtra, India.
E-mail: vandana2609@gmail.com
ABSTRACT
Ultra performance liquid chromatography (UPLC) takes advantage of technological strides made in particle chemistry performance, system
optimization, detector design and data processing and control. Using sub-2 mm particles and mobile phases at high linear velocities and
instrumentation that operates at higher pressures than those used in HPLC, dramatic increases in resolution, sensitivity and speed of analysis
can be obtained. This new category of analytical separation science retains the practicality and principles of HPLC while creating a step
function improvement in chromatographic performance. This review introduces the theory of UPLC and summarizes some of the most recent
work in the field.
KEYWORDS: UPLC, HPLC, Resolution, Sensitivity.
INTRODUCTION
UPLC refers to Ultra Performance Liquid
Chromatography. It improves in three areas:
chromatographic resolution, speed and sensitivity
analysis. It uses fine particles and saves time and
reduces solvent consumption. UPLC is comes from
HPLC. HPLC has been the evolution of the packing
materials used to effect the separation. An underlying
principle of HPLC dictates that as column packing
particle size decreases, efficiency and thus resolution
also increases. As particle size decreases to less than
2.5µm, there is a significant gain in efficiency and it
doesn’t diminish at increased linear velocities or flow
rates according to the common Van Deemter equation.
By using smaller particles, speed and peak capacity
(number of peaks resolved per unit time) can be
extended to new limits which is known as Ultra
Performance. The classic separation method is of HPLC
(High Performance Liquid Chromatography) with many
advantages like robustness, ease of use, good selectivity
and adjustable sensitivity. It’s main limitation is the lack
of efficiency compared to gas chromatography or the
capillary electrophoresis due to low diffusion
coefficients in liquid phase, involving slow diffusion of
analytes in the stationary phase.1,2
The Van Deemter
equation shows that efficiency increases with the use of
smaller size particles but this leads to a rapid increase in
back pressure, while most of the HPLC system can
operate only up to 400 bar.3
That is why short columns
filled with particles of about 2µm are used with these
systems, to accelerate the analysis without loss of
efficiency, while maintaining an acceptable loss of load.
To improve the efficiency of HPLC separations, the
following can be done:-
i) Work at higher temperatures
ii) Use of monolithic columns
USE OF THE UPLC SYSTEM
Elevated-temperature chromatography also allows for
high flow rates by lowering the viscosity of the mobile
phase, which significantly reduces the column back
pressure. Monolithic columns contain a polymerized
porous support structure that provides lower flow
resistances than conventional particle-packed
columns.4,5,6
PRINCIPLE
The UPLC is based on the principal 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). The Van Deemter curve,
governed by an equation with three components shows
that the usable flow range for a good efficiency with
small diameter particles is much greater than for larger
2. Patil V. P. et al. IRJP 2 (6) 2011 39-44
IRJP 2 (6) June 2011 Page 39-44
diameters.7,8
H=A+B/v+Cv
Where A, B and C are constants and v is the linear
velocity, the carrier gas flow rate. The A term is
independent of velocity and represents "eddy" mixing. It
is smallest when the packed column particles are small
and uniform. The B term represents axial diffusion or
the natural diffusion tendency of molecules. This effect
is diminished at high flow rates and so this term is
divided by v. The C term is due to kinetic resistance to
equilibrium in the separation process. The kinetic
resistance is the time lag involved in moving from the
gas phase to the packing stationary phase and back
again. The greater the flow of gas, the more a molecule
on the packing tends to lag behind molecules in the
mobile phase. Thus this term is proportional to v.
Therefore it is possible to increase throughput and thus
the speed of analysis without affecting the
chromatographic performance. The advent of UPLC has
demanded the development of a new instrumental
system for liquid chromatography, which can take
advantage of the separation performance (by reducing
dead volumes) and consistent with the pressures (about
8000 to 15,000 PSI, compared with 2500 to 5000 PSI in
HPLC). Efficiency is proportional to column length and
inversely proportional to the particle size18. Therefore,
the column can be shortened by the same factor as the
particle size without loss of resolution. Efficiency is
three times greater with 1.7 μm particles compared to 5
μm particles and two times greater compared to 3.5 μm
particles. Resolution is 70% higher than with 5 μm
particles and 40% higher than with 3.5 μm particles.
High speed is obtained because column length with 1.7
μm particles can be reduced by a factor of 3 compared to
5 μm particles for the same efficiency and flow rate can
be three times higher. The application of UPLC resulted
in the detection of additional drug metabolites, superior
separation and improved spectral quality.9,10
SMALL PARTICLE CHEMISTRY
The promises of the van Deemter equation cannot be
fulfilled without smaller particles than those
traditionally used in HPLC. The design and
development of sub-2 mm particles is a significant
challenge and researchers have been active in this area
for some time to capitalize on their advantages.
Although high efficiency, non-porous 1.5 mm particles
are commercially available, they suffer from poor
loading capacity and retention due to low surface area.
To maintain retention and capacity similar to HPLC,
UPLC must use novel porous particles that can
withstand high pressures. Silica based particles have
good mechanical strength, but can suffer from a number
of disadvantages, which include a limited pH range and
tailing of basic analytes. Polymeric columns can
overcome pH limitations, but they have their own issues,
including low efficiencies and limited capacities. In
2000, generation hybrid chemistry that took advantage
of the best of both the silica and polymeric column
worlds was introduced. Produced using a classical sol-
gel synthesis that incorporates carbon in the form of
methyl groups, these columns are mechanically strong
with high efficiency and operate over an extended pH
range. But, in order to provide the kind of enhanced
mechanical stability required for UPLC, a second
generation bridged ethane hybrid (BEH) technology was
developed. These 1.7 mm particles derive their enhanced
mechanical stability by bridging the methyl groups in
the silica matrix.
Packing 1.7 mm particles into reproducible and rugged
columns was also a challenge that needed to be
overcome. Requirements include a smoother interior
surface of the column hardware, and re-designing the
end frits to retain the small particles and resist clogging.
Packed bed uniformity is also critical, especially if
shorter columns are to maintain resolution while
accomplishing the goal of faster separations. In addition,
at high pressures, frictional heating of the mobile phase
can be quite significant and must be considered. With
column diameters typically used in HPLC (3.0 to 4.6
mm), a consequence of frictional heating is the loss of
performance due to temperature induced non uniform.
To minimize the effects of frictional heating, smaller
diameter columns (1–2.1 mm) are typically used for
UPLC.11
INSTRUMENTATION
Sample injection
In UPLC, sample introduction is critical. Conventional
injection valves, either automated or manual, are not
designed and hardened to work at extreme pressure. To
protect the column from extreme pressure fluctuations,
the injection process must be relatively pulse-free and
the swept volume of the device also needs to be minimal
to reduce potential band spreading. A fast injection
cycle time is needed to fully capitalize on the speed
afforded by UPLC, which in turn requires a high sample
capacity. Low volume injections with minimal carryover
are also required to increase sensitivity. There are also
direct injection approaches for biological samples.12,13
UPLC Column
Resolution is increased in a 1.7 µm particle packed
column because efficiency is better. Separation of the
components of a sample requires a bonded phase that
provides both retention and selectivity. Four bonded
phases are available for UPLC separations: ACQUITY
3. Patil V. P. et al. IRJP 2 (6) 2011 39-44
IRJP 2 (6) June 2011 Page 39-44
UPLCT M BEH C18 and C8 (straight chain alkyl
columns), ACQUITY UPLC BEH Shield RP18
(embedded polar group column) and ACQUITY UPLC
BEH Phenyl (phenyl group tethered to the silyl
functionality with a C6 alkyl). Each column chemistry
provides a different combination of hydrophobicity,
silanol activity, hydrolytic stability and chemical
interaction with analytes.
ACQUITY UPLC BEH C18 and C8 columns are
considered the universal columns of choice for most
UPLC separations by providing the widest pH range.
They incorporate trifunctional ligand bonding
chemistries which produce superior low pH stability.
This low pH stability is combined with the high pH
stability of the 1.7 µm BEH particle to deliver the widest
usable pH operating range. ACQUITY UPLC BEH
Shield RP18 columns are designed to provide
selectivities that complement the ACQUITY UPLC
BEH C18 and C8 phases. ACQUITY UPLC BEH
Phenyl columns utilize a trifunctional C6 alkyl tether
between the phenyl ring and the silyl functionality. This
ligand, combined with the same proprietary end capping
processes as the ACQUITY UPLC BEH C18 and C8
columns, provides long column lifetimes and excellent
peak shape. This unique combination of ligand and end
capping on the 1.7 µm BEH particle creates a new
dimension in selectivity allowing a quick match to the
existing HPLC column. An internal dimension (ID) of
2.1 mm column is used. For maximum resolution,
choose a 100 mm length and for faster analysis, and
higher sample throughput, choose 50 mm column. Half-
height peak widths of less than one second are obtained
with 1.7µm particles, which gives significant challenges
for the detector. In order to integrate an analyte peak
accurately and reproducibly, the detector sampling rate
must be high enough to capture enough data points
across the peak. The detector cell must have minimal
dispersion (volume) to preserve separation efficiency.
Conceptually, the sensitivity increase for UPLC
detection should be 2-3 times higher than HPLC
separations, depending on the detection technique. MS
detection is significantly enhanced by UPLC; an
increased peak concentration with reduced
chromatographic dispersion at lower flow rates
promotes increased source ionization efficiencies. The
ACQUITY UPLC System consists of a binary solvent
manager, sample manager including the column heater,
detector, and optional sample organizer. The binary
solvent manager uses two individual serial flow pumps
to deliver a parallel binary gradient. There are built-in
solvent select valves to choose from up to four solvents.
There is a 15,000-psi pressure limit (about 1000 bar) to
take full advantage of the sub-2µm particles. The sample
manager also incorporates several technology
advancements. Using pressure assisted sample
introduction, low dispersion is maintained through the
injection process, and a series of pressures transducers
facilitate self-monitoring and diagnostics. It uses needle-
in-needle sampling for improved ruggedness and needle
calibration sensor increases accuracy. Injection cycle
time is 25 seconds without a wash and 60 sec with a
dual wash used to further decrease carry over. A variety
of micro titer plate formats (deep well, mid height, or
vials) can also be accommodated in a thermostatically
controlled environment. Using the optional sample
organizer, the sample manager can inject from up to 22
micro titer plates. The sample manager also controls the
column heater. Column temperatures up to 65°C can be
attained. To minimize sample dispersion, a “pivot out”
design allows the column outlet to be placed in closer
proximity to the source inlet of an MS detector.14
For UPLC detection, the tunable UV/Visible detector is
used which includes new electronics and firmware to
support Ethernet communications at the high data rates.
Conventional absorbance-based optical detectors are
concentration sensitive detectors, and for UPLC use, the
flow cell volume would have to be reduced in standard
UV/Visible detectors to maintain concentration and
signal. According to Beer’s Law, smaller volume
conventional flow cells would also reduce the path
length upon which the signal strength depends. A
reduction in cross-section means the light path is
reduced, and transmission drops with increasing noise.
Therefore, if a conventional HPLC flow cell were used,
UPLC sensitivity would be compromised. The
ACQUITY Tunable UV/Visible detector cell consists of
a light guided flow cell equivalent to an optical fiber.
Light is efficiently transferred down the flow cell in an
internal reflectance mode that still maintains a 10mm
flow cell path length with a volume of only 500 ml.
Tubing and connections in the system are efficiently
routed to maintain low dispersion and to take advantage
of leak detectors that interact with the software to alert
the user to potential problems.15
ADVANTAGES
· Decreases run time and increases sensitivity.
· Provides the selectivity, sensitivity and dynamic
range of LC analysis.
· Maintaining resolution performance.
· Expands scope of Multiresidue Methods.
· UPLC’s fast resolving power quickly quantifies
related and unrelated compounds.
· Faster analysis through the use of a novel separation
material of very fine particle size.
4. Patil V. P. et al. IRJP 2 (6) 2011 39-44
IRJP 2 (6) June 2011 Page 39-44
· Operation cost is reduced.
· Less solvent consumption.
· Reduces process cycle times, so that more product
can be produced with existing resources.
· Increases sample throughput and enables
manufacturers to produce more material that
consistently meet or exceeds the product
specifications, potentially eliminating variability,
failed batches or the need to re-work material.
· Delivers real-time analysis in step with
manufacturing processes.
· Assures end-product quality, including final release
testing. 17
DISADVANTAGES
Due to increased pressure requires more maintenance
and reduces the life of the columns of this type. So far
performance similar or even higher has been
demonstrated by using stationary phases of size around
2 µm without the adverse effects of high pressure. In
addition, the phases of less than 2 µm are generally non-
regenerable and thus have limited use.
APPLICATIONS OF UPLC
Analysis of amino acids
UPLC used for accurate, reliable and reproducible
analysis of amino acids in the area of protein
characterization, cell culture monitoring and nutritional
analysis of foods.
Analysis of natural products and traditional herbal
medicine
UPLC is widely used for analysis of natural products and
herbal medicines. The main purpose of this is to analyze
drug samples arise from the complexity of the matrix and
variability from sample to sample. Purification and
qualitative and quantitative chromatography and mass
spectrometry are being applied to determine active drug
candidates and to characterize the efficacy of their
candidate remedies. UPLC provides high-quality
separations and detection capabilities to identify active
compounds in highly complex samples that results from
natural products and traditional herbal medicines.
Analysis of Levofloxacin in human plasma.
Identification of Metabolite
Biotransformation of new chemical entities (NCE) is
necessary for drug discovery. When a compound
reaches the development stage, metabolite identification
becomes a regulated process. It is of the utmost
importance for lab to successfully detect and identify all
circulating metabolites of a candidate drug. Discovery
studies are generally carried out in vitro to identify
major metabolites so that metabolic weak spots on the
drug candidate molecule can be recognized and
protected by changing the compound structure.
Study of Metabonomics / Metabolomics
Metabonomics studies are carried out in labs to
accelerate the development of new medicines. The
ability to compare and contrast large sample groups
provides insight into the biochemical changes that occur
when a biological system is exposed to a new chemical
entity (NCE). Metabonomics provides a rapid and robust
method for detecting these changes improves
understanding of potential toxicity and allows
monitoring the efficacy.
ADME Screening
ADME studies measure physical and biochemical
properties absorption, distribution, metabolism,
elimination, and toxicity of drugs where such
compounds exhibit activity against the target disease. A
significant number of candidate medicines fall out of the
development process due to toxicity. If toxic reactions
or any side effect occurs in the discovery/development
process, then it becomes more costly. It is difficult to
evaluate candidate drugs for possible toxicity, drug-drug
interactions, inhibition, and/or induction of metabolizing
enzymes in the body. Failure to properly identify these
potential toxic events can cause a compound to be
withdrawn from the market. The high resolution of
UPLC enables accurate detection and integration of
peaks in complex matrices and extra sensitivity allows
peak detection for samples generated by lower
concentration incubations and sample pooling.
Bioanalysis/ Bioequivalence Studies
For pharmacokinetic, toxicity, and bioequivalence
studies, quantization of a drug in biological samples is
an important part of development programs. The drugs
are generally of low molecular weight and are tested
during both preclinical and clinical studies. Several
biological matrices are used for quantitative
bioanalysis, the most common being blood, plasma and
urine.
Dissolution Testing
For quality control and release in drug manufacturing,
dissolution testing is essential in the formulation,
development and production process. In sustained-release
dosage formulations, testing higher potency drugs is
particularly important where dissolution can be the rate-
limiting step in medicine delivery. The dissolution profile
is used to demonstrate reliability and batch-to-batch
uniformity of the active ingredient. Additionally, newer
and more potent formulations require increased analytical
sensitivity.
Forced Degradation Studies
One of the most important factors that impacts the quality
and safety of pharmaceuticals is chemical stability. The
FDA and ICH require stability testing data to understand
5. Patil V. P. et al. IRJP 2 (6) 2011 39-44
IRJP 2 (6) June 2011 Page 39-44
how the quality of an API (active pharmaceutical
ingredient) or a drug product changes with time under the
influence of environmental factors such as heat, light,
pressure and moisture or humidity. Knowledge of these
stability characteristics defines storage conditions and
shelf life, the selection of proper formulations and
protective packaging and is required for regulatory
documentation. Forced degradation or stress testing is
carried out under even harsher conditions than those used
for accelerated stability testing.
Manufacturing/ QA/ QC
Identity, purity, quality, safety and efficacy are the
important factors to be considered while manufacturing
a drug product. The successful production of quality
pharmaceutical products requires that raw materials meet
purity specifications. That manufacturing processes
proceed as designed. That final pharmaceutical product
meets, and hopefully exceeds, defined release
specifications. Continued monitoring of material stability
is also a component of quality assurance and control.
UPLC is used for the highly regulated, quantitative
analyses performed in QA/QC laboratories.
Method Development / Validation
According to FDA, validation is defined as establishing
documented evidence that provides a high degree of
assurance that a specific process will consistently produce
a product meeting its predetermined specifications and
quality attributes. Method development and validation is a
time-consuming and complicated process: labs need to
evaluate multiple combinations of mobile phase, pH,
temperature, column chemistries and gradient profiles to
arrive at a robust, reliable separation for every activity.
UPLC help in critical laboratory function by increasing
efficiency, reducing costs and improving opportunities for
business.
Impurity Profiling
For the drug development and formulation process,
profiling, detecting, and quantifying drug substances and
their impurities in raw materials and final product
testing is an essential part. Impurity profiling requires
high-resolution chromatography capable of reliably and
reproducibly separating and detecting all of the known
impurities of the active compound. Also critical is the
ability to accurately measure low-level impurities at the
same time as the higher concentration active
pharmaceutical component. UPLC System and Columns
specifically address high-throughput analysis
requirements while maintaining high peak resolution.
UPLC also involves the latest peak detection algorithms
and custom calculations to optimize data processing and
reporting. It also confidently detects impurities in
compounds even at trace levels.
Compound Library Maintenance
Confirming the identity and purity of a candidate
pharmaceutical is critical to effectively screening
chemical libraries that contain vast types of small
molecules across a range of biological targets. Chemists
need to be sure they have synthesized the expected
compound. In this high-throughput screening
environment, the ability to obtain information in
multiple MS and UV detection modes in a single
injection is invaluable Combining fast analysis with
open-access software delivers the power of LC/MS to
chemists who are not analytical instrumentation
specialists. A single complete system enables them
thoroughly screen a compound, from sample
introduction to end results.
Open Access
Maximum efficiency is essential for analytical
laboratories that are constantly challenged to increase
throughput and deliver results to research chemists in
pharmaceutical discovery. UPLC and UPLC/MS
systems and software enable versatile and open
operation for medicinal chemistry labs, with easy-to-use
instruments, a user-friendly software interface, and fast,
robust analyses using UV or MS for nominal and exact
mass measurements. 18,19,20
CONCLUSION
UPLC increases productivity in both chemistry and
instrumentation by providing more information per unit of
work as it gives increased resolution, speed, and
sensitivity for liquid chromatography. The main
advantage is a reduction of analysis time, which also
meant reduced solvent consumption. The time spent
optimizing new methods can also be greatly reduced. The
time needed for column equilibration while using gradient
elution and during method validation is much shorter.
Sensitivity can be compared by studying the peak width at
half height. It was found that the sensitivity of UPLC was
much higher than that of conventional HPLC.
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Table 1: Comparison between UPLC and HPLC16
Characteristics HPLC Assay UPLC Assay
Column 150 X 3.2 mm 150 X 2.1 mm
Particle size 3 to 5mm Less than 2mm
Flow rate 3.0 ml / min 0.6 ml / min
Needle wash Methanol Methanol
Injection volume 5µL (Std.In 100 % MeOH) 2µL(Std.In 100 % MeOH)
Column temperature 30 °C 65 °C
Maximum backpressure 35-40 MPa 103.5 MPa
Gradient (time in min) ACN:H2O
T0 (25:75), T6.5 (25:75), T7.5 (95:5), T9
(25:75), T10 (25:75)
T0 (36:64), T1.1 (95:5), T1.3 (36:64)
Total run time 10min 1.5min
Total solvent consumption (including 0.5
min of delay time in between injections)
Acetonitrile: 10.5 ml
Water: 21.0 ml
Acetonitrile: 0.53 ml
Water: 0.66 ml
Plate count 2000 7500
USP resolution 3.2 3.4
Delay volume 750 μl 110 μl