Government College University Faisalabad

Assignment
Topic: Ultra Performance Liquid
Chromatography (UPLC)
Subject: Advanced Pharmaceutical Analysis
Submitted to: Dr. Sajid Akash
Submitted by: Qudsia Rehman 606

1
 Ultra Performance Liquid Chromatography (UPLC)

1. Introduction
UPLC refers to Ultra Performance Liquid Chromatography or Ultra Pressure Liquid
Chromatography. It is new category of analytical separation science works on the similar
principles of HPLC while increasing the overall interlaced attributes of speed, sensitivity &
resolution. It uses fine particles and saves time and reduces solvent consumption. UPLC is
a rising chromatographic separation technique whose packing materials have smaller
particle size lesser than 2.5μm.The technology takes full advantage of chromatographic
principles to run separations using columns packed with smaller particles and higher flow
rates.
2. Principle
The UPLC is based on the principle 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 that
describes the relationship between linear velocity (flow rate) and plate height (HETP or
column efficiency).
H=A+B/v + Cv
Where,
A = Eddy diffusion. It is smallest when the packed column particles are small and uniform.
B = Natural diffusion tendency of molecules. This effect is diminished at high flow rates
and so this term is divided by v.
C = Represents the kinetic resistance to equilibrium during the process of separation.
v = flow rate
3. Chemistry of Small Size Particles
The chemistry of the particles used in this course of the method contributes the increased
efficiency and potential to work at amplified linear velocity, thereby, providing both the
speed and the resolution.
Resolution can be described as

2
 Where, α represents the selectivity factor, N is efficiency and K denotes proportionality
constant. According to this, resolution increases with increase in efficiency. Since
efficiency (N) is inversely proportional to particle size (dp)
N α L/dp
Therefore, the length of the column (L) may be reduced by the same ratio as the size of the
particle without losing the resolution.
Efficiency also has indirect relationship with the peak width (w) according to the equation:
N α 1/w2
This means that narrower the peaks are, easier is their separation from each other. Peak
height (H) also has opposite relationship with peak width (w) according to equation:
H α 1/w
The effect of particle size on HETP and linear velocity has been illustrated in graph by
using van Deemter plot which shows that the particles with a smaller diameter are
contributing less to band broadening compared to larger particles and are less affected by
higher column flow rate.

Van Deemter Plot illustrating the effect of particle size (in µm) on plate height (H). Smaller
particle size provides higher overall peak efficiencies and a much wide range of flow rates.
4. Instrumentation
i. Pumping System
ii. Sample injection

3
 iii. UPLC columns
iv. Column Heater
v. Detectors

i. Pumping System
The calculated pressure drop at the optimum flow rate for maximum efficiency across
a 15 cm long column packed with 1.7 μm particles is about 15,000 psi. Pumps are
capable of delivering solvent smoothly and reproducibly at this pressure.
There are two types of pumps:
i. Reciprocating pump
ii. Pneumatic pump
Requirements for standard UPLC pump
 Volume of sample injection is less as 3-5 micro liters
 Pump operates at 1000 psi pressure
 Particle size in stationary phase packing material is less than 2 micro liter
ii. Sample injection
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.
iii. UPLC Columns
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:
1) ACQUITY UPLCTM BEH C18 & C8
These are considered as 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

4
 combined with the high pH stability of the 1.7μm BEH particle to deliver the widest
usable pH operating range.
2) ACQUITY UPLC BEH Shield RP18 (embedded polar group column): These
are designed to provide selectivity's that complement the ACQUITY UPLC
BEHTM C18 and C8 Columns.
3) ACQUITY UPLC BEH phenyl columns: These utilize a trifunctional C6 alkyl
ethyl between the phenyl ring.
4) ACQUITY UPLC BEH Amide columns:
o BEH particle technology, in combination with a trifunctionally bonded amide
phase, provides exceptional column life time, thus improving assay robustness.
o BEH Amide columns facilitate the use of a wide range of phase pH

iv. Column Heater

The column heater heats the column compartment to any temperature from 50°C to
65°C.
5. Detectors
Mainly three types of detectors are employed in UPLC:

5
 d
o
w
f
P
T
s
a
E
I
tS
D
b
U
9
1
(
A
.u
m
0
l5
r
a
g
p
y
in
c
r
v
h
e
V
7
)
n
t
h
C
L
/u
μ
2
,z
T
6. Comparison between HPLC and UPLC

7. Advantages


Decreases run time and increases sensitivity.
Reducing analysis time so that more products can be produced with existing
resources.

6
  Provides the selectivity, sensitivity, and dynamic range of LC analysis
 Maintains resolution performance.
 Fast resolving power quickly quantifies related and unrelated compounds.
 Operation cost is reduced.
 Less solvent consumption

8. Disadvantages
 Due to increased pressure requires more maintenance and reduces the life of the
columns of this type.
 The phases of less than 2 μm are generally non-regenerable and thus have limited use.

9. Applications
 Analysis of natural products and traditional herbal medicine.
 Identification of metabolite.
 Study of metabonomics/metabolomics.
 Bio analysis/bioequivalence studies.
 Manufacturing/QA/QC
UPLC is used as an important tool in QA/QC laboratories for the quantitative and
extremely regulated analysis.
 Impurity profiling
The presence of excipients in the sample makes the profiling difficult and with
HPLC method, it takes longer time for analysis to achieve sufficient resolution.
Thus, the combination of UPLC with mass spectrometry has been useful for the
documentation of drug and endogenous metabolites in the final product.
 Identification of Metabolic Biomarkers to Diagnose Epithelial Ovarian Cancer
(EOC)
Currently available tests are insufficient to distinguish patients with EOC from
normal individuals. Plasma specimens of EOC patients and normal individuals were
analyzed using UPLC/MS. Eight biomarkers were identified which may serve as
novel biomarkers for diagnosis.The application of UPLC in the diagnosis of EOC is
shown by the graph below:

7
8