UPLC

ULTRA PERFORMANCE LIQUID

CHROMAROGRAPHY
 CONTENT

• INTRODUCTION
• PRINCIPLE
• INSTRUMENTATION
• BASIC DIFF. IN HPLC AND UPLC
• ADVANTAGES
• DISADVANTAGES
• Factor affect performance of UPLC
• APPLICATIONS
• REFERENCE
 INTRODUCTION

• “It is separation technique involving mass transfer between stationary phase

and mobile phase”
• Separated species appeared as colored bands on columns.
• 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
 INTRODUCTION

• Special analytical columns UPLC BEH C18 packed with 1.7µm particles are
used in connection with system
• The factor responsible for development of UPLC technique was evolution
of packing material used to effect the separation.
• The technology takes full advantage of chromatographic principles to run
separations using columns packed with smaller particles.
• It decreases analysis time and solvent consumption.
 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
 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
 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.
 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
 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
 INSTRUMENTATION

• Columns
• 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
 INSTRUMENTATION

Columns
BEH phenyl columns:
• Phenyl group attach with C6 alkyl.

• Each column chemistry provides different combination of hydrophobicity,

hydrolytic stability and chemical interaction with analyte
 INSTRUMENTATION

• Detectors

• Tunable ultra violet detector

• Fluorescence Detector
• Refractive-Index Detector
• Evaporative Light Scattering Detector
BASIC DIFFERENCE
Chromatograms of Simvastatin
 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 residuel method.
• Reduces process cycle time.
• Less solvent consumption .
 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
FACTORS AFFECTING PRFORMANCE OF UPLC

• Pressure
• Column
• Particle size of packing
• Temperature
 FACTORS AFFECTING PRFORMANCE OF UPLC

FIGURE 1- Van Deemter plot illustrating the evolution of

particle sizes over the last three decades.
 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
 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
 REFERENCE

• 1. Chatwal G. Anand S. Instrumental Methods of Chemical Analysis. 5th edition, Himalaya publication
house, 2006, 2.6912.699.
• 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
• 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
•Thaank you