ULTRA PERFORMANCE

LIQUID
CHROMATOGRAPHY(UPLC)
PRESENTED BY
K.HEMA HARINI
(169V1R0033)
 CONTENTS

❖INTRODUCTION
❖COMPARISON BETWEEN UPLC AND HPLC
❖INSTRUMENTATION
❖ADVANTAGES & DISADVANTAGES OF UPLC
❖APPLICATIONS OF UPLC
 Introduction
• UPLC refers to Ultra Performance Liquid Chromatography, which is a modern
technique that gives a new direction for liquid chromatography i.e. it focuses &
enhances mainly in three areas: “speed, resolution and sensitivity.
• Ultra performance liquid chromatography (UPLC) is applicable for particles less
than 2µm in diameter to acquire better resolution, speed, and sensitivity.
• The separation and quantification in UPLC is done under very high pressure (up to
100m pa). As compare to HPLC, under high pressure it is observed that it does not
have any negative influence on analytical column and also other components like
time and solvent consumption.
 COMPARISON BETWEEN UPLC & HPLC
UPLC HPLC

• More selective and sensitive. • Less selective and sensitive.

• High resolving power. • Low resolving power.

• Particle size of stationary phase is less than 2 • Particle size of stationary phase is between 3 to
micron. 5 microns.
• It decreases the consumption of solvent and • More solvent consumption, less sample
increases sample throughput. throughput.
• It requires high pressure, decreases the life of • It requires low pressure, increases the life of
column. column.
• It has narrow use. • It has more use.

• Detectors use small flow rates and low • Detectors use high flow rates and high
detection volume. detection volume.
• Injection volume is small, less run time. • Injection volume is large, more run time.
 INSTRUMENTATION
➢ Instrumentation mainly includes
A) Sample injections
B)UPLC columns
C) Detectors
❑SAMPLE INJECTIONS:

In UPLC, sample introduction is critical. 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 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.
❑ UPLC COLUMNS:
• Commonly used bond phases for retention and selectivity are:
(i) ACQUITY UPLCTM BEH C8 (straight chain alkyl columns):These columns are considered as
universal columns for most of UPLC separations by providing wide range of pH .
(ii) ACQUITY UPLCTM BEH C18 (straight chain alkyl columns):These columns are designed to provide
selectivity that complements the ACQUITYUPLC BEH C18 and C8 phases.
(iii) ACQUITY UPLC BEH Shield RP18 (embedded polar group column): These columns utilize a tri
functional C6 alkyl tether between the phenyl ring and the silyl functionality, , provides long column lifetimes
and excellent peak shape.
(iv) ACQUITY UPLC BEH Phenyl (phenyl group tethered to the silyl functionality with a C6 alkyl)
ACQUITY UPLC BEH Phenyl (phenyl group tethered to the silyl functionality with a C6 alkyl).
• 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 in-built solvent select valves to choose from up to four solvents.
• 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.
 DETECTORS
• The detectors used in UPLC analysis is UV/visible
detector. Detection of analytes is conventionally based on
absorbance i.e.concentration sensitivity detectors. In
UPLC the flow cell volume should be reduced to
maintain concentration and signal. 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 500ml. 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 .
 ADVANTAGES&DISADVANTAGES OF UPLC
ADVANTAGES OF UPLC:
➢ Require less run time and enhance sensitivity.
➢ In chromatogram resolved peaks are obtained.
➢ Multi residue methods are applied.
➢ Speedy analysis, quantify accurately analytes and related products.
➢ Uses of fine particle (2μm) for packing of stationary phase make analysis fast.
➢ Time and cost both are reduced..Consumption of solvents is less.
➢ More products are analyzed with existing resources.
DISADVANTAGES OF UPLC:
➢ In UPLC analysis the main disadvantage occurs are life of columns, during analysis high pressure developed
because of the particle size.
➢ Increase pressure reduces the life of the columns. Due to increased pressure more maintenance is required .
 APPLICATIONS OF UPLC
✓ In pharmaceutical industry the demand of UPLC analysis is very high, because of the unique features of UPLC
like high resolution in chromatogram, short time analysis which make more analytical work in less time with
valuable, reliable and authentic data.
✓ Scientist can generate more accurate data by UPLC in faster way. UPLC technique is used for the analysis of
herbal product.

✓ By this method the standard of analysis in every aspect like qualitative, quantitative and complexity of sample
can be differentiate in very high standard . It is very useful in bio-analytical field.
✓ The unique features of UPLC i.e. high resolution and speedy analysis also very helpful in pharmacokinetic
studies like – adsorption, distribution, metabolism and excretion (ADME).