ULTRA HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC): A NEW TREND IN ANALYSIS

Abstract

UPLC is an advance technique of liquid chromatography where it takes advantage of innovation in various technologies such as instrumentation and particle size to achieve dramatic increases in resolution, speed and sensitivity of the liquid chromatography. It operates at higher pressure than that used in HPLC and uses fine particles (less than 2.5μm) & mobile phases at high linear velocities. UPLC Technology is now applied throughout the world produce quality data with reproducible and robust methods as compared to the conventional HPLC. UPLC can be hyphenated with other techniques such as Mass spectrometer (MS), Ion chromatograph (IC), Nuclear magnetic resonance spectrometer (NMR) and Infrared spectrometer (IR) etc. This technique provides unique end-to-end solutions for all industries and has found application in various fields such as pharmaceutical, food, environmental, forensic, toxicology and pesticide.

Full text

www.wjpr.net Vol 5, Issue 3, 2016.
387
Shaikh et al. World Journal of Pharmaceutical Research
ULTRA HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
(UPLC): A NEW TREND IN ANALYSIS
Dr. Sayyed Hussain and Tabrez Shaikh*
Sir Sayyed College, P.G. Department of Chemistry, Roshangate, P.B.No.89, Aurangabad,
India.
ABSTRACT
UPLC is an advance technique of liquid chromatography where it takes
advantage of innovation in various technologies such as
instrumentation and particle size to achieve dramatic increases in
resolution, speed and sensitivity of the liquid chromatography. It
operates at higher pressure than that used in HPLC and uses fine
particles (less than 2.5µm) & mobile phases at high linear velocities.
UPLC Technology is now applied throughout the world produce
quality data with reproducible and robust methods as compared to the
conventional HPLC. UPLC can be hyphenated with other techniques
such as Mass spectrometer (MS), Ion chromatograph (IC), Nuclear
magnetic resonance spectrometer (NMR) and Infrared spectrometer
(IR) etc. This technique provides unique end-to-end solutions for all industries and has found
application in various fields such as pharmaceutical, food, environmental, forensic,
toxicology and pesticide.
KEYWORD: UPLC, Theory, Detectors, Advantage, Applications.
INTRODUCTION
From past 30 year HPLC is the predominant technique to use for analysis in Laboratory but
due to significant advances and innovation in instrumentation, detector design, data
processing and particle size technology, leads to the development of Ultra Performance
Liquid Chromatography (UPLC). Principle of UPLC basically remains same only with the
help of technology; it achieves dramatic increases in resolution, speed and sensitivity of the
liquid chromatography. UPLC Technology is applied throughout the world for providing
World Journal of Pharmaceutical Research
SJIF Impact Factor 6.805
Volume 5, Issue 3, 387-394. Review Article ISSN 2277– 7105
Article Received on
22 Dec 2015,
Revised on 13 Jan 2016,
Accepted on 03 Feb 2016
*Correspondence for
Author
Tabrez Shaikh
Sir Sayyed College, P.G.
Department of Chemistry,
Roshangate, P.B.No.89,
Aurangabad, India.

www.wjpr.net Vol 5, Issue 3, 2016.
388
Shaikh et al. World Journal of Pharmaceutical Research
advantage of time, cost and quality. Beside this it is also reproducible result and robust
method as compared to conventional HPLC.
THEORY
Separation in chromatography can be explained by Van Deemter equation which gives
relation of resolving power of chromatographic column with various flows and Kinetic mass
transfer.[1]
HETP = A + B/u + Cu,
Where,
HETP is height equivalent to theoretical plate
A is eddy diffusion
B is longitudinal diffusion
C is mass transfer and
u is linear velocity (flow rate)
Eddy diffusion (A) is caused by a turbulence in the solute flow path and is mainly unaffected
by flow rate, but changes with particle size. Smaller the particle size less is the eddy
diffusion. Longitudinal diffusion (B) is related the movement of an analyte molecule outward
from the center to the edges of its band. Thus higher the lineary velocities will limit the
outward distribution, keeping the band tighter. Mass transfer (C) is the movement of analyte,
or transfer of its mass, between the mobile and stationary phases. Through this type of
diffusion, increased flows have been observed to widen analyte bands or lower peak
efficiencies. [2]
Fig. 1 - Van Deemter plots for various particle sizes

www.wjpr.net Vol 5, Issue 3, 2016.
389
Shaikh et al. World Journal of Pharmaceutical Research
Fig-1 indicates that the decrease in particle size results in an increase in efficiency of column
and on the other hand increase in linear velocity (flow rate) increase the efficiency for the
column for particle size less than 1.9 µm and after the optimized flow it remains same, while
for column with particle size greater than 1.9µm, efficiency again decrease after certain
optimized flow. [3]
Therefore, using smaller particles, speed and peak capacity (number of peaks resolved per
unit time) can be extended to new limits also decrease in the internal diameter of the column
will require a less flow rate which is known as Ultra Performance. Larger diameter columns
require higher flow rates, and thus larger volumes of mobile phase, to reach the desired linear
velocity therefore UPLC have column with less diameter.
INSTRUMENTATION
The Basic principle and instrumentation of UPLC system remains same as that of HPLC but
only differs in upgrading instrumentation and hardware. The UPLC System consists of a
binary solvent system, sample manager, column manager and detector. The solvent manager
uses two flow pumps to deliver a parallel binary gradient which is mixed under high pressure.
[4] Degassing system degasses the mobile phase, which is selected by valve up to four solvent.
UPLC system can withstand pressure of about 15,000 psi (about 1000 bar) to take full
advantage of the sub-2-mm particles.[5] The sample manager is also having advance
technology where sample temperature can be taken to as low as 0°C [6] whereas column
manger can manage the column temperature up to 90°C using high temperature ultra
performance liquid chromatography (UPLC), it is possible to drastically decrease the analysis
time without loss in efficiency.[7]
Fig. 2 – Schematic diagram of UPLC

www.wjpr.net Vol 5, Issue 3, 2016.
390
Shaikh et al. World Journal of Pharmaceutical Research
DETECTOR AND HYPENATION OF UPLC
Detectors such as UV/Visible, Photodiode array (PDA), Evaporative light scattering (ELS),
Refractive index (RI) and Fluorescence (FLR) are commonly used with UPLC. Beside these
detectors, capability of UPLC can be greatly increased by hyphenating instrument with other
technique such as mass spectrometer, ion chromatograph, nuclear magnetic resonance
spectrometer, inductive coupled plasma-mass spectrometer and Infrared spectrometer.
Ultra-violet/visible (UV)
This detector are used for organic compound which absorb the light in the range of 190 to
800 nm This detector can be tuned to specific wavelengths in UV or Visible range for
detection. It provides performance benefits for both routine and complex analyses in
pharmaceutical, life science, environmental, agricultural and petrochemical applications.
Photodiode Array (PDA) detector
This detector offers simultaneous advanced optical detection in the range of 190 to 800 nm. It
provides unprecedented trace impurity detection and quantitation with spectral analysis
capabilities. Definitive compound identification and co-elution detection with simultaneous
2D and 3D operation. This detector finds major application in drug discovery and
pharmaceutical development.
Fluorescence (FLR) Detector
For sensitivity and selectivity to fluorescence-based applications, this detector is used. It
extends the benefits of UPLC technology for the analysis of polynuclear aromatic
hydrocarbons (PAHs), drugs of abuse, and vitamins – any component with chemiluminescent
properties, such as fluorescence or phosphorescence.
Refractive index (RI) detector
RI is a universal detector which is used where chemical is having no or limited UV
absorbance. These include alcohols, sugars, fatty acids, excipents, raw material and
pharmaceutical drug products. Beside this characterization of low molecular weight polymers
also finds application on UPLC. Main disadvantage of this detector is that it lacks sensitivity.
Mass detector (MS)
UPLC can be coupled with mass spectrometrer (MS) and tandem mass spectrometer (MS-
MS) detector which find application in various fields and is used for identification,

www.wjpr.net Vol 5, Issue 3, 2016.
391
Shaikh et al. World Journal of Pharmaceutical Research
quantitation and mass analysis of materials. Also, structural elucidation of unknown molecule
can be found out by fragmentation. This detector have various mass analyzers depending
upon their application some of analyzer are Single quadrupole, Triple quadrupoles (Tandem),
Ion trap and Time of flight (TOF). These detectors provide very high sensitivity, selectivity
and time resolution.
Beside these detector many other detectors can be hyphenated to UPLC such as Infrared (IR),
Inductive Coupled plasma mass spectrometry (ICP-MS), nuclear magnetic resonance (NMR)
and Evaporative light scattering detector (ELSD), Electrochemical detector (EC)
ADVANTAGE OF UPLC OVER HPLC
UPLC has many advantages over conventional HPLC of which major advantages are speed,
quality and cost of analysis. Due to development of particle size technology and the use of
sub micron particle size in column packing, significantly reduced in the analysis run time
which result in the development of faster methods and faster analysis of samples. Compound
separated on UPLC are more resolved as compared to conventional HPLC also peak capacity
is also increased (number of peaks resolved per unit time). Sensitivity of the method is
increased up to 3- 5 fold. Band spreading is reduced due to the population of analyte
molecule is more concentrated in UPLC resulting in a higher plate count. Following figure-3
show the comparison of UPLC and HPLC chromatogram.
Fig. 3 – Chromatographic separation comparison on UPLC and HPLC

www.wjpr.net Vol 5, Issue 3, 2016.
392
Shaikh et al. World Journal of Pharmaceutical Research
The time spent in optimizing and validating new methods is greatly reduced with the use of
UPLC. Mobile phase solvent consumption for the analysis is greatly reduced due to low flow
rate and short run time. Reduces process cycle times, so that more product can be produced
with existing resources. Real time analysis by UPLC reduced the cost of failure of product
and process control. UPLC system can be hyphenated with various techniques for which it
finds application in vast areas. Conventional HPLC are nowadays are replaced with UPLC
considering greater commercial benefit, superior sensitivity, resolution, speed and sample
throughput.
APPLICATIONS OF UPLC
Pharmaceutical analysis
UPLC finds major application in pharmaceutical analysis. Methods use in drug substances
and drug product analysis should be well developed and validated and these processes are
much time consuming on conventional HPLC. UPLC gives scope for development and
validation of analytical methods in less time. In the drug development impurity profiling is
major activity where detecting and quantifying impurities in drug substance and drug product
can be done by UPLC as it has good resolving power, reproducibility, efficiency and short
time. As UPLC provides faster analysis of samples, this advantage can be use to monitor the
inprocess and real time samples of reaction monitoring where very short time is required to
control the processes thus saving the cost of failure. UPLC also finds application in
dissolution testing, which is one of very important test in the formulation of drug product.
This test requires uniformity reliability and batch to batch reproducibility. Metabolites studies
are required in the new drug development process where main metabolite is determined and
identified as rapidly as possible in the drug discovery phase. UPLC is capable of determining
and identifying metabolite and biomarker structure elucidation.
Environmental analysis
Environmental sample requires innovative techniques to detect and identify the chemical
contamination. UPLC provides the analysis of these samples with less time, cost and more
information about sample content. Some applications of UPLC are analysis of organic
component in soil, air, hazardous wastes, drinking water, wastewater, pesticide residue and
perfluorinated compounds (PFCs) analysis.

www.wjpr.net Vol 5, Issue 3, 2016.
393
Shaikh et al. World Journal of Pharmaceutical Research
Food analysis
Food manufacturer are always in search of a compressive solution for food testing thus UPLC
decrease operation cost, increase productivity and provide identification of diverse chemicals
in a food sample, thus providing public safety. Beside this it also offers quality and
consistency of the product. UPLC is also applied for food profiling, identification of natural
toxins, vitamins and pesticide residue in food product.
Forensic and Toxicological
UPLC finds good application in the identification of drug of abuse from blood, urine and oral
samples. Several cannabinoids, opioids, barbiturates can be identified and analysed by
UPLC. The combination of UPLC with various instruments gives the unique benefit of drug
screening with excellent sensitivity and accuracy at trace level.
CONCLUSION
UPLC is a powerful emerging technique which by use of advances in instrumentation and
particle technology increases the productivity for pharmaceutical and other industries. This
technique provides the better resolution for separating component, high sensitivity for the
analysis of low concentration component and reduces time of analysis. As solvent
consumption for this technique is less it also reduces the cost of analysis. The method can be
well developed and validated in less time on this system. This technique can be easily
hyphenated with various other techniques such as mass spectrometry, UPLC finds versatile
application for impurity profiling, metabolite identification, dissolution testing and process
control analysis in pharmaceutical, food, environment, forensic and Toxicological areas.
REFERENCES
1. Swartz, Michael E. "UPLC™: an introduction and review." Journal of Liquid
Chromatography & Related Technologies 2005; 28(7-8): 1253-1263
2. Zhang, Bin, Xiaofeng Li, and Bing Yan. "Advances in HPLC detection - towards
universal detection." Analytical and bioanalytical chemistry 2008; 390(1): 299-301.
3. Wren, Stephen AC, and Pierre Tchelitcheff. "Use of ultra-performance liquid
chromatography in pharmaceutical development." Journal of Chromatography A 2006;
1119(1): 140-146.
4. Swartz, Michael E., and Brian Murphy. "New frontiers in chromatography." Am. Lab
2005; 37: 22-27.

www.wjpr.net Vol 5, Issue 3, 2016.
394
Shaikh et al. World Journal of Pharmaceutical Research
5. Wu, Naijun, and Richard Thompson. "Fast and efficient separations using reversed phase
liquid chromatography." Journal of liquid chromatography & related technologies 2006;
29(7-8): 949-988.
6. Wales, Thomas E., et al. "High-speed and high-resolution UPLC separation at zero
degrees Celsius." Analytical chemistry 2008; 80.17: 6815-6820.
7. Nguyen, Dao T-T., et al. "High throughput liquid chromatography with sub-2μm particles
at high pressure and high temperature." Journal of Chromatography A 2007; 1167.1:
76-84.