This document provides an overview of ultra performance liquid chromatography (UPLC). It begins with an introduction to chromatography and defines UPLC. The main advantages of UPLC are listed as decreased run time, increased sensitivity, and maintaining resolution. The document outlines the basic principles, instrumentation, and differences between UPLC and HPLC. Applications discussed include drug discovery, herbal medicine analysis, and metabolomics studies. The document is presented by Mr. Atish Khilari and provides references for additional information.

1. ULTRA PERFORMANCE LIQUID
CHROMAROGRAPHY
Presented by
Mr. Atish Khilari
M. Pharm. 1st sem
(2016-2017)
Guided By
Prof. Mr. M. T. Mohite
Department of pharmaceutics
Dr. D. Y. Patil College of Pharmacy, Akurdi,Pune-44

2. CONTENT
INTRODUCTION
ADVANTAGES
DISADVANTAGES
PRINCIPLE
INSTRUMENTATION
BASIC DIFF. IN HPLC AND UPLC
APPLICATIONS
REFERENCE

3. INTRODUCTION
CHROMATOGRAPHY-
(Chromo-color, logos-writing)
 “It is separation technique involving
mass transfer between stationary phase
and mobile phase”
Separated species appeared as colored
bands on columns.

4. INTRODUCTION
UPLC-
It refers “Ultra Performance Liquid
Chromatography”.
Which used in analytical techniques.
It improves three areas-Speed, resolution,
sensitivity.
Speed-1.5min
Pressure-15000psi
Sensitivity-3-5µl

5. ADVANTAGES
Decreases run time and increases sensitivity.
Provides the selectivity, sensitivity, and
Maintaining resolution performance.
UPLC’s fast resolving power quickly
quantifies related and unrelated compounds.
Use of multi residue method.
Reduces process cycle time.
Less solvent consumption .

6. DISADVANTAGES
Due to increased pressure requires more
maintenance and Reduces the life of the
columns.
The phases of less than 2μm are generally
non-regenerable.

7. PRINCIPLE
Principle of UPLC is based on Van-Deemter
equation which describes relationship
between flow rate and HETP or column
efficiency.
H=A+B/µ+Cµ
Where,
A is related to the mobile phase movement
through paths in the stationary phase.
B describes longitudinal diffusion.
C relates the analyte to mass transfer between
the pores of the stationary phase

8. INSTRUMENTATION

9. INSTRUMENTATION
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.

10. INSTRUMENTATION
Pump-
 Constant pressure pump-
 constant pressure is used only for column
packing. .
 Constant flow pump-This type is mostly used
in all common UPLC application.
 Reciprocating piston pump.
 Dual piston pump.

11. INSTRUMENTATION
Sample injection system-
 To protect the column from extreme back
pressure fluctuations.
 The injection system must be relatively pulse
free and fast injection cycle time is needed
 thats why low volume injection system are used
which can deliver 3-5 microltr sample.
 Low volume injection also require to increase
sensitivity.

12. INSTRUMENTATION
Columns-
 Paricle size of packing material is
approximately 1.7 μm.
 Which increase speed of separation and
efficiency of column.
 Separation of the component of sample require
bonded phase that provide both selectivity and
retension.
That bonded phases are-

13. INSTRUMENTATION
ACQUITY UPLC BEH C18 AND C8 columns-
 These are straight alkyl chains.
 Columns were designed to be the universal
columns of choice for most UPLC
separation by providing widest pH range.
ACQUITY UPLC BEH Shield RP18-
 Columns are designed to provide selectivities
that complement the ACQUITY UPLC BEH C18
and C8 phases.

14. INSTRUMENTATION
 Unique bonding chemistry provides
complementary selectivity and enhances the
peak shape for basic compounds and
 Yielding compatibility with 100% aqueous mobile
phases.
BEH phenyl columns:
 Phenyl group attach with C6 alkyl.
 Each column chemistry provides different
combination of hydrophobicity, hydrolytic
stability and chemical interaction with analyte.

15. INSTRUMENTATION
Detectors-
Tunable ultra violet detectors-
UV detectors are most frequently used to
measure components showing an absorption
spectrum in the ultraviolet or visible region.
A UV detector uses deuterium discharge lamp
(D2 lamp) as a light source, with the wavelength of
its light ranging from 190 to 380 nm

16. INSTRUMENTATION

17. INSTRUMENTATION
Fluorescence Detector

18. INSTRUMENTATION
 Most pharmaceuticals, natural products, clinical
samples, and petroleum products have
fluorescent absorbance.
 For some compounds which do not have
fluorescence absorbance or low absorbance,
they can be treated with fluorescence derivatives
such as dansylchloride.
 The system is easy to operate and relatively
stable.

19. Refractive-Index Detector
RI detector measures change
in reflex index.
INSTRUMENTATION

20. INSTRUMENTATION
 Evaporative Light Scattering Detector-
ELSD provides good sensitivity for non-
volatile analytes.
↓
The column effluent is nebulized and then
evaporated to make it form fine particles.
↓
The analyte is then radiated with a laser beam
and the scattered radiation is detected.
↓
The target sample includes lipids, sugar, and
high molecular weight analytes.

21. BASIC DIFFERENCE
Characteristics HPLC UPLC
Speed 10 min. 1.5 min.
Pressure 6000PSi 15000PSi
Particle size >4 µm Appr.1.7µm
Injection Volume 20 µl 3-5 µl
Colum dimension 150 ×3.2 mm 150× 2.1 mm
Analytical column C8 and C18 Bonded phase column
are used.(UPLC BEH
C18)

22. Chromatograms of Simvastatin
Lc system-
ACQUITY UPLC SYSTEM.
Column-Xbridge.
4.6×250mm
M.ph-65:35 acetonitrile phosphate buffer.
Flow rate-1.5ml/min
Inj.vol-10 microltr
Lc system-
ACQUITY UPLC SYSTEM
Column-ACQUITY UPLC BEH C18
2.1×100mm
M.ph-65:35 acetonitrile aq.buffer
Flow rate-0.56ml/min
Inj.vol-0.8 microltr

23. APPLICATIONS
Drug Discovery -UPLC improves the drug
discovery process by means of high
throughput screening, combinational
chemistry.
Analysis of natural products and herbal
traditional medicines.
Analysis of Dosage form- It provides
high speed, accuracy and reproducible
results for analysis of drugs and their related
substance. Thus method development time
decrease.

24. APPLICATIONS
 Analysis of amino acids-
UPLC used for accurate, reliable and
reproducible analysis of amino acids in area of
protein characterisation, cell culture monitoring
and nutritional analysis of food.
 Study of metabonomics/ metabolomics-
 Metabonomics studies are carried out in labs to
accelerate development of new medicines.

25. REFERENCE
1. Chatwal G. Anand S. Instrumental
Methods of Chemical Analysis. 5th
edition,
Himalaya publication house, 2006, 2.691-
2.699.
2. Kasture A., Wadodkar S.,
Pharmaceutical Analysis, 6th edition, Vol-2,
Nirali Prakashan, 2005, 22-26.
3.Jerkovitch AD, Mellors JS, and Jorgenson
JW. LCGC North America, 2003;7.
4. Lippert JA, and Lee ML. J. Chromotogr.
2001; 911: 1.

26. REFERENCE
5 T. Sunil Kumar Reddy, G. Balammal,
International journal of pharmaceutical research
and analysis, UPLC: An introduction and review,
Vol. 2 , Issue 1,2012,24-31
6. Patil V.P,Tathe R.D,T.,UPLC-A Review, IRJP
2(6), 2011,39-44.

27. THANK
YOU