Ultra Performance Liquid

Chromatography

Presented by

Bindiya Patel

Agenda

• Definition• Introduction• Principal• Advantages• Disadvantages• Instrumentation• Application

Objective

• To focus on ultra performance liquid chromatography

technique.

• To study its instrumentation.

• To know its various application.

What is chromatography ???

• Chromatographic process can be defined as

separation technique involving mass transfer

between stationary and mobile phase.

• The separated species appeared

as a colored bands on the column,

which account for the name for

the method,

In Greek chromo meaning “color”,

Graphic meaning “writing”

Introduction• UPLC refers to Ultra Performance Liquid Chromatography.

• UPLC is one mode of chromatography, used in analytical techniques.

• It improves in three areas: chromatographic resolution, speed and

sensitivity.

• 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.

Principle• The principle of UPLC is based on Van Deemter equation which describes

the relationship between flow rate and HETP or column efficiency

H=A+B/v + Cv

Where,

A = Eddy diffusion

B = Longitudinal diffusion

C = Equilibrium mass transfer

v = flow rate

van Deemter equation, that describes the relationship between linear

velocity (flow rate) and plate height (HETP or column efficiency)

Advantages…• Decreases run time and increases sensitivity.

• Reducing analysis time so that more product

can be produced with existing resources.

• 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.

Disadvantages

• Due to increased pressure requires more maintenance 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.

Comparison between HPLC and UPLC

CHARACTERISTICS HPLC UPLC

Particle size 3-5μm Less than 2μm

Maximum back pressure35-40 Mpa

less103.5 Mpa

more

Column Alltima C18Acquity UPLC BEH

C18

Column dimension 150 X 3.2 mm 150 X 2.1 mm

Column temperature 30 °C 65 °C

Volume injection 5μL (Std. In100% MeOH) 2μL (Std.In100% MeOH)

Sample throughput less more

Sample preparation simple tedious

Column coagulation Does not takes place Takes place

Analysis time more less

Sensitivity less higher

UPLC Instrument

ation

Instrumentation

1. Pumping systems

Achieving small particle, high peak capacity separations 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-2μm particles.

Cont…2. Sample injection In UPLC, 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 potential band spreading.

Low volume injections with minimal carryover

required to increase sensitivity.

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 the silyl functionality

with a C6 alkyl)

4. ACQUITY UPLC BEH Amide columns (trifunctionally bonded amide phase).

Cont…• ACQUITY UPLCTM BEH C18 & C8 .

1. These are considered as the universal columns of choice for most

UPLC separations by providing the widest pH range.

2. They incorporate trifunctional ligand bonding chemistries which

produce superior low pH stability.

3. This low pH stability is combined with the high pH stability of the

1.7μm BEH particle to deliver the widest usable pH operating range

Cont…• Acquity UPLC BEH Shield RP18

These are designed to provide selectivity's that complement the ACQUITY UPLC BEH T M C18 and C8 Columns.

• Acquity UPLC BEH phenyl columns These utilize a trifunctional C6 alkyl ethyl between the phenyl ring.• Acquity UPLC BEH Amide columns1. BEH particle technology, in combination with a trifunctionally bonded amide phase, provides exceptional column life time, thus improving assay robustness. 2. BEH Amide columns facilitate the use of a wide range of phase pH [2 –11].

Acquity UPLC BEH Column chemistries

Detectors• Detectors used are1. UV detectors2. Fluorescent detector3. Refractive index detector4. Light scattering detector5. Electrochemical detector6. Mass spectrometric detector

Phototube Consist of high sensitive cathode in a form of

half cylinder in evacuated tube.

Anode is also present along the axis of the

tube.

Inside layer is coated with light sensitive layer.

When light is incident surface coating emits

electron this is attracted and collected by

anode.

Current which is created between cathode

and anode is regarded as measure of radiation

falling on the detector.

Photomultiplier Ejected photoelectron strikes dynode,

secondary e- released

Voltage accelerates e- to next dynode and so

Result is large charge packet

hitting anode

High Gain & detected

Fluorescence detector The light from an excitation source passes through a

filter or monochromator, and strikes the sample.

A proportion of the incident light is absorbed by the

sample, and some of the molecules in the sample

fluoresce. The fluorescent light is emitted in all

directions.

Some of this fluorescent light passes through a

second filter or monochromator and reaches a

detector, which is usually placed at 90° to the incident

light beam to minimize the risk of transmitted or

reflected incident light reaching the detector.

Evaporative light scattering detector

 This detector based on the deflection principle of refractometry,

where the deflection of a light beam is changed when the composition

in the sample flow-cell changes in relation to the reference side (as

eluting sample moves through the system).

As sample elutes through one side, the changing

angle of refraction moves the beam.

This results in a change in the photon current

falling on the detector which unbalances it.

The extent of unbalance (which can be related

to the sample concentration) is recorded on a

strip chart recorder.

Refractive index detector

Common UPLC chromatographic conditions1. Columns: ACQUITY UPLC BEM C18, BEH Shield RP18, BEH C8 OR BEH

Phenyl Column.2. Dimensions: 2.1 X 50mm 1.7μm.3. Mobile Phase A1: 20mM NH4COOH in H2O, pH 3.0.

4. Mobile Phase A2: 20mM NH4HCOOH in H2O, pH 10.0.

5. Mobile Phase B1: Acetonitrile .6. Mobile Phase B2: Methanol. 7. Flow rate: 0.5ml/min. 8. Injection Volume: 10.0μl. 9. Week needle wash: 3% methanol. 10. Strong needle wash: 90% acetonitrile.11. Temperature: 30°C. 12. Detection: UV 254 nm. 13. Sampling rate: 20pts/sec.

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.