UPLC provides faster, more sensitive and efficient separations compared to HPLC by using sub-2 μm particles. It operates at higher pressures of up to 100 MPa. Smaller particle sizes and columns allow for shorter run times, lower detection limits, less solvent usage and improved resolution. Key aspects of UPLC instrumentation include high-pressure binary pumps, low-volume injection systems, 1.7 μm particle columns, and detectors adapted for smaller flow cells like tunable UV detectors. UPLC finds applications in pharmaceutical analysis, natural products analysis, and metabolomics studies.

1. ULTRA PERFOEMENCE
LIQUID CHROMATOGRAPHY
PRESENTED BY :- BHAGYASRI SANJAYRAO PATIL
[ M.PHARM 1ST SEM, PHARMACEUTICAL CHEMISTRY]

2. CONTENTS :-
 Definition
 Introduction
 Principle
 Comparison between HPLC & UPLC
 Instrumentation
 Advantages
 Disadvantage
 Application

3. DEFINITION :-
• Chromatographic process can be defined as separation technique involving mass
transfer between stationary and mobile phase.
• The separation species appeared as a coloured bands on the column, which account for the
name for the method.

4. INTRODUCTION :-
• UPLC is a modern technique which gives a new direction for liquid chromatography.
• UPLC refers to ultra performance liquid chromatography, which enhance mainly in three
areas: “speed, resolution and sensitivity.
• Ultra performance liquid chromatography (UPLC) application for particle less than
2𝜇𝑚 in diameter to acquire better resolution, speed, and sensitivity compared with high-
performance liquid chromatography*(HPLC).
• In 21st centuary pharmaceutical industries are focousing for new ways to in economy and
shorten time for development of drugs.

5. CONT…….
• 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 not any negative influence
on analytica; column and also other components like time and solvent consumption is less
in UPLC.

6. PRINCIPLE :-
• High performance liquid chromatography (HPLC) has proven to one of the most and
predominant technology used in analytical laboratories for the analysis of drugs
worldwide during the past 30 - plus years.
• One of the basic concerns for the growth of this technique is the packing material which
effects the separations.
• In this separation mechanism the principle apply is van deemeter equation , with which
any student of chromatography is intimately familiar.

7. VAN DEEMETER EQUATION
H = A + B/v + Cv
Where,
 A = Eddy diffusion
 B = Longitudinal diffusion
 C = Equilibrium mass transfer
 v = flow rate
Van deemeter equation, that describes the relationship between linear velocity (flow rate) and plate height (column
efficiency) And, since particle size is one od he variables, a Van Deemeter curve can be used to investigate
chromatographic performance.

8. COMPARISON BETWEEN HPLC & UPLC
UPLC
1. Particle size od stationary phase is less than 2 𝜇𝑚
2. Operates at higher pressure due to reduced particle size
maximum back pressure of 1000 bars /100 Mpa
3. Inner diameter of the column is generally 0.75-1.8mm
4. More selective and sensitive
5. High resolving power
6. Reduce process cycle time and assures end-product
quality with reduced cost of operation and decreased
run time
7. It decrease the consumption of solvent and increases
sample throughput
HPLC
1. Particle size of stationary phase is between
3 to 5 𝜇𝑚
2. Operates at relatively lower pressure than
UPLC maximum backpressure between
300-400 bars
3. Inner diameter of the column is 3-10 mm
4. Less selective and sensitive
5. Less resolving power
6. High cost of operation and high run time
as compared to UPLC
7. More solvent consumption, less sample
throughput

9. CONT……
8. Low extra column dispersion
9. Higher precision in sample
introduction.
10. Detector that uses small flow rates and
flow detection limits.
8. More extra column band
broadening
9. Lower precision comparative
in sample introduction.
10. Detector that uses large flow
rates and larger detection cells.

10. INSTRUMENTATION:-
 1. Pumping system
 2. Sample Injection
 3. UPLC column
 4. Detector

12. 1. PUMPING SYSTEM :-
 Achieving small particle, high peak capacity separation requires a greater pressure range.
 Both the gradient and isocratic separation modes are used.
 the binary solvent manager uses two individual serial flow pumps to deliver a parallel
binary gradient.
 There are built-in solvent select valves to choose from up to four solvents.
 There is a 15,000 – psi pressure limit ( about 1000 bar) to take full advantage of the sub
2 𝜇𝑚 particles.

13. 2. SAMPLE INJECTION :-
 In UPLC, sample introduction is critical. Conventional injection valve either automated
or manual, are not designed and hardend 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 potential band spreading.
 Low volume injections with minimal carryover required to increase sensitivity.

14. 3. 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 (straight chain alkyl columns)
2. ACQUITY UPLC BEH Shield RP18 ( embedded polar group column )
3. ACQUITY UPLC BEH Phenyl ( Phenyl group tethered to thr silyl functionality with a C6 alkyl)
4. ACQUITY UPLC BEH Amide columns (trifunctionally bonded amide phase).

15. 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 combient with high pH stability of the 1.7 𝜇𝑚 BEH particle to
deliver the widest usable pH operating range.

16. 2. ACQUITY UPLC BEH SHIELD RP18
• These are designed to provide selectivity’s that compement the ACQUITY UPLCTM BEH C18
& 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
• BEH Amide columns facilitate the use of a trifunctional column life time, thus
improving assay robustness.
• BEH Amide columns facilitate the use of a wide range of phase pH (2 – 11 ).

17. ACQUITY UPLC BEH
Column chemistries

18. DETECTORS :-
UV DETECTOR
• In the uv detector detection of analytes is conventionally based on absorbance that is concentration
sensitivity detectors.
• In UHPLC the flow cell volume would have to be reduced to maintain concentration and signal.
• It based on Beer’s law, smaller volume conventional flow cells would also reduce the path length upon
which the signal strength depends.
• A reduction in cross-section means the light path is reduced, and transmission drops with increasing noise.
• Therefore, if a conventional HPLC flow cell were used, UPLC sensitivity would be cpmpeomised .
• The ACQUITY Tunable UV/visible detector cells consists of a light guided flow cell equivalent to a
optical fiber.

19. CONT……
• Light is efficiently transferred down the flow cell in an internal reflectance mode that 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 detector that interact with software to alert the user potential
problems.

20. ADVANTAGES OF UPLC
• Decrease run time and increases sensitivity.
• Reducing analysis time so that more product can be produced with existing resources.
• Provides the selectivity, sensitivity, and duanamic range of LC analysis
• Maintains resolution performance.
• Fast resolving power quickly quantifires related and unrelated compounds.
• Operation cost is reduced.
• Less solvent consumption.

21. DISADVANTAGES OF UPLC
• Due to increased pressure required more maintance and reduces the life of the columns of
this type.
• In addition, the phases of less than 2 𝜇𝑚 are generally non-regenerable and thus have
limited use.

22. APPLICATION
• Analysis of natural products and traditional herbal medicine.
• Identification of metabolite.
• Study of metabonomics / metabolomics.
• Bio analysis/ bioequivalence studies.
• Manufacturing / QA / QC
• Impurity profiling.
• Forced degradation studies.
• Dissolution Testing.
• Toxicity Studies.