ULTRA PERFORMANCE

LIQUID
CHROMATOGRAPHY
UNDER THE GUIDANCE OF: PRESENTED BY:
[Link] SRUTHI [Link]
[Link], Ph.D 2024MPH40009
IPT-SPMVV [Link],I YEAR, II SEM
PHARMACEUTICAL
ANALYSIS
 Table of contents

01 02 03

INTRODUCTION PRINCIPLE INSTRUMENTATION

04 05 06

ADVANTAGES& APPLICATIONS COMPARISION

DISADVANTAGES
 01
INTRODUCTION
 Ultra High Performance Liquid Chromatography (UHPLC) Modern Liquid
Chromatographic system -(Modification of HPLC).

 Developed by Waters corporation (2004).

 UHPLC is similar to HPLC technique, used to separate different

constituents of a compound.

 Used to identify, quantify and separate components of a mixture by using

High pressure to push Solvents through the Column.
 02
PRINCIPLE
 In this separation mechanism the principle apply is Van Deemter equation,

H=A+B/ μ +C μ

H= Height equivalent to theoritical plates

μ= Average linear velocity of the mobile phase

A= Eddy diffusion

B= Longitudinal diffusion

C= Mass transfer
THEORETICAL PLATES:
It is an imaginary or hypothetical unit of the columns where distribution of solute
between stationary phase and mobile phase has attained equilibrium.
• These can also be called as functional unit of the column.

solute molecules

Stationary Mobile
phase phase
1)Number of theoretical plates:

o Efficiency of column is expressed by theoretical plate.

o It can be determined by using following formula;

N= 16(tR/Wb)^2

Where tR= Retention time

Wb= Peak width

o If number of theoretical plates is high then the column said to be more

efficient
2) Height equivalent to theoretical plate(HETP):

 Plate height is the distance in which solute moves in one portion of

the column.

 If HETP is less then column is more efficient and vice versa

 It can be determined by using the formula;

HETP=l/n

where l= Length of the column

n= Number of theoretical plates

I. Eddy diffusion (A, multiple path effect):

• A term is independent of velocity and represents eddy mixing.

• If the column is not uniformly packed then unequal path lengths may
exist in the column, some solute molecules of same species may travel
short path and get eluted fast while other molecules may travel long path
(more distance) and get eluted late.
II. Axial diffusion(B, longitudinal diffusion):

 As the band of solute molecules travel in the mobile phase, it will tend
to diffuse in all directions (in less concentrated region).
 If mobile phase velocity is low then high diffusion rates of solute in
the mobile phase can cause the solute molecules to disperse axially
and due this they will be eluted slowly from column.
 To minimise longitudinal diffusion mobile phase flow rate should be
optimum(high)
III)Mass transfer (Cs and Cm):

 The analyte takes a certain amount of time to equilibrate between

the stationary and mobile phase.
 Term Cs results from resistance to mass transfer at the solute to
stationary phase interface.
 Cs is directly proportional to effective thickness of the stationary
phase (df) and inversely proportional to diffusion coefficient of the
solute in stationary phase
 Slow molecular movements within the stationary phase means a
longer time spent in this phase by the solute molecules, while other
molecules are moving forward with mobile phase.
 If mobile phase velocity is high and mass transfer rate is slow then
solute band will be broad.
IV)Average liner velocity of the mobile phase (μ) –

 At low velocity of mobile phase longitudinal diffusion will be more and at

high velocity of mobile phase Cs (resistance to mass transfer from
stationary phase) will be more.
 Both these will cause band broadening.
 To obtain efficient separation mobile phase velocity should be optimum.
 03
INSTRUMENTATION
COMPONENTS:

1. Solvent reservoir

2. Pump

3. Sample injector

4. UPLC Column

5. Detector
1. SOLVENT RESERVOIR:

 The most common type of solvent reservoir is glass bottle.

 Most of manufacturers supply these bottles with special caps, tubing and filters to
connect to the pump inlet and so the purge gas (Helium) used to
remove dissolved air.
2. PUMP:

 The UHPLC pump is responsible for delivering the mobile phase at a

constant flow rate and pressure to the chromatographic column.
 It is designed to handle high pressures, typically up to 15,000 psi,
enabling faster and more efficient separations compared to traditional
HPLC systems.
 These are of two types;
a. Isocratic pump
b. Gradient pump
3. SAMPLE INJECTION :

 In UHPLC, 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 the potential band spreading.
 Low volume injections with minimal carryover required to increase sensitivity.
Fig: Load and inject position of the sample.
[Link]:
 UHPLC column is a cylindrical tube that contains a stationary phase, which
is responsible for the separation of different compounds in sample.
 It is designed to have a small particle size and a high packing density, which
allows for faster and more efficient separation compared to traditional HPLC
columns.
 The small particle provides a larger surface area for interactions between the
sample components and the stationary phase, resulting in improved
resolution.
 There are different types of UHPLC columns, such as reversed-phase,
normal-phase and ion-exchange columns, each with its own specific
applications.
Ex: ACQUITY UPLCTM BEH C18
5. DETECTOR:

 UHPLC detectors are designed to detect and measure the analytes based on
their physical or chemical properties.
 There are several types of detectors commonly used in UHPLC :-
- UV detector
- Fluorescent detector
- Refractive index detector
- Light scattering detector
- Electrochemical detector
- Mass spectroscopic detector
 04
ADVANTAGES & DISADVANTAGES
ADVANTAGES:

 Decreases run time and increases sensitivity.

 Faster analysis through the use of a novel separation material of very fine
particle size.
 Operation cost is reduced.
 Less solvent consumption.

DISADVANTAGES:

 Due to increased pressure requires more maintenance and reduce the life
of the column.
 The phase of less than 2 µm are generally non-regenerable thus have
limited use.
 05
APPLICATIONS
APPLICATIONS:

 Analysis of natural products & herbal medicines.

 Analysis of amino acids.
 Identification of metabolites(Metabolomic studies).
 Method development and validation.
 Manufacturing & QA/QC.
 Detection & identification of impurities.
 Stability testing.
 Dissolution testing.
 Pharmacokinetic & bioequivalence studies.
 Screening of synthetic compounds.
 Food analysis.
 Toxicity studies.
 06
COMPARISION
S. UHPLC HPLC
No.

1. Particle size of stationary phase is Particle size of stationary phase is

<2µ(typically 1.7µ) between 3-5µ.
2. Operates at higher pressure Operates at relatively lower pressure
(15,000psi)due to reduced particle (6,000psi)than UHPLC
size
3. Maximum back pressure of Maximum back pressure between 300-
1000bars/100MPa 400 bars
4. Inner diameter of column is 0.75- Inner diameter of column is 3-10mm
1.8mm
5. More selective and sensitive Less selective and sensitive

6. High resolving power. Less resolving power.

S. UHPLC HPLC
No.
7. It decreases the consumption of the More solvent consumption, less sample
solvent and increases the sample throughput
throughput
8. Detectors uses small flow rates and Detector uses higher flow rates and high
low detection volume. detector volume.
9. Injection volume is small(2µl). Injection volume is large(5µl).

10. More plate count is achieved i.e. Less plate count is achieved i.e.2,000
75,000
11. Column used-AQUITY UHPLC Column used-Xterra, C18,50×4.6mm
BEH C18,50×2.1mm
12. Total run time- 1.5min Total run time- 10 min
REFERENCES:

1. Taleuzzaman [Link] S. Ultra Performance Liquid Chromatography-A

[Link] of Analytical and pharmaceutical chemistry.2015;2(6):1056.

2. Dong MW, Zhang K. Ultra-high-pressure liquid chromatography (UHPLC) in

method development. TrAC Trends in Analytical Chemistry. 2014 Dec 1;63:21-30.

3. Chawla G, Ranjan C. Principle, Instrumentation and Applications of UPLC: A

Novel Technique of Liquid Chromatography. Open Chem J. 2016;3(1):1–16.
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