INSTRUMENTATION OF UPLC

AND TYPES OF COLUMNS USED.

NAME- BHAVANA RAJENDRA PANDHARE.

STD- Msc PART- II, SEM III
PAPER-I 1
 INTRODUCTION……..
• Ultra Performance Liquid
Chromatography is a type of liquid
chromatography techniques used to
separate the different components
found in mixtures.

• UPLC is a special version of HPLC

which has the advantage of
technological strides made in particle
chemistry performance, system
optimization, detector design, data
processing and control.
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 INTRODUCTION……..
• UHPLC provides the separation
science with high speed, high
resolution, high sensitivity, and short
runtime.

• Ultra-high pressure liquid

chromatographic system can
withstand extremely high pressures,
up to 1,000 bars, which a sub-2 m
particle column generates.

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DIFFERENCE ……..

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 PRINCIPLE……..
 The underlying principle of UPLC is based on the van Deemter relationship
which explains the correlation between flow rate and plate height.

 Where A, B and C are constants and v is the flow rate, the carrier gas flow rate.
A= Eddy diffusion
B = longitudinal diffusion
C= non equilibrum Solute’s mass transfer
H=height equivalent to the theoretical plate

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 PRINCIPLE……..
(A) Eddy diffusion: (B)Longitudinal Diffusion:
 Independent of flow rate  Tendency of molecule to flow from
higher concentration to lower
concentration.

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 PRINCIPLE……..
(C) Non equilibrium mass transfer : Optimum
condition
for flow
rate

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 MOBILE PHASE……..
 Mobile phases mobile phases mainly consist of organic solvent and buffer.
Acetonitrile and methanol are commonly used organic solvents.
Tetrahydrofuran (THF) may also be used as a UPLC organic solvent.

 The other component of mobile phase is aqueous buffer.

The buffer pH of a mobile phase should be outside pka ± 2 to ensure method
robustness.
The most commonly used mobile phase buffer is a trifluoroacetic acid (TFA)
aqueous solution.

 Different buffer pH are investigated during method development for acidic and
basic compounds.

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INSTRUMENTATION ……..
 Parts of UPLC are as follows:

A. Sample Injection
B. Solvent reservoir
C. High pressure pump
D. UPLC Columns
E. Detectors

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 INSTRUMENTATION ……..
A. Sample Injection:

 In UPLC, sample introduction is critical.

 Either automated or manual.
 The injection must be done reproducibly and
accurately.
 The volume of the sample in UPLC is usually 2-5 μl.
 Direct injection approaches are utilized for the
biological samples.
 Low volume injections with minimal carryover are
also required to increase sensitivity.

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 INSTRUMENTATION ……..
B. Solvent reservoir:

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

 Most of the manufactuers 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.

C. High pressure pump:

 Constant pressure pumps-

 Constant flow pumps-
 Reciprocating piston pumps-
 Dual piston pumps-
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 INSTRUMENTATION ……..
D. UPLC Columns:

 Coreshell HPLC/UHPLC columns of high efficiency and performance. Different

types of columns being used in UPLC are packed with particles which are
produced through different technologies.

 These are as follows:

1. Charged Surface Hybrid [CSH] particle technology

2. Ethylene Bridged Hybrid [BEH] particle technology
3. High Strength Silica [HSS] particle technology and
4. Peptide Separation Technology (PST)

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 TYPES OF
COLUMN

ACQUITY ACQUITY ACQUITY ACQUITY

UPLCTM BEH UPLCTM BEH UPLC BEH UPLC BEH
C18 C8 phenyl shield RP18

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 TYPES OF COLUMNS USED……..
(A) ACQUITY UPLCTM BEH C8 AND ACQUITY UPLCTM BEH C18 :

 straight chain alkyl columns

 universal columns
 provides the widest pH range
 They incorporate tri functional ligand bonding chemistries ACQUITY UPLCTM BEH C8
which produce superior low pH stability.
 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.

ACQUITY UPLCTM BEH C18

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 TYPES OF COLUMNS USED……..
(B) ACQUITY UPLC BEH Shield RP18 :

 embedded polar group column

 designed to provide selectivity that complements the
ACQUITY UPLC BEH C18 and C8 phases.

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 TYPES OF COLUMNS USED……..
(C) ACQUITY UPLC BEH Phenyl:

 phenyl group tethered to the silyl functionality with a C6

alkyl
 This ligand, combined with the same proprietary end
capping processes as the ACQUITY UPLC BEH C18 and C8
columns, provides long column lifetimes and excellent peak
shape.
 This unique combination of ligand and end capping on the
1.7μm BEH particle creates a new dimension in selectivity
allowing a quick match to the existing HPLC column.

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INSTRUMENTATION ……..
E. Detectors:

 UV/Visible detector
 Fluoresent detectors
 Refractive index detectors
 Light scattering detectors
 Electrochemical detectors
 Mass spectrometric detector

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ADVANTAGES……..
 It is more selective and sensitive

 high resolution performance and faster resolving power.

 It also reduces process cycle time and assures end-product quality with reduced cost of
operation and decreased run time.

 It increases sensitivity.

 It decreases the consumption of solvent

 increases sample throughput

 provides real-time analysis in step with manufacturing processes. 20

 DISADVANTAGES……..
 The higher back pressures compared to conventional HPLC.

 More maintenance

 Increasing the column temperature.

 Moreover, the particles of less than 2 μm are mostly non-regenerable and, therefore,
have a narrow use.

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 REFERENCES ……..
 Taleuzzaman M, Ali S, Gilani SJ, Imam SS and Hafeez A. Ultra Performance Liquid
Chromatography (UPLC) - A Review. Austin J Anal Pharm Chem. 2015; 2(6): 1056.

 Gita Chawla and Chanda Ranjan, Principle, Instrumentation, and Applications of

UPLC: A Novel Technique of Liquid Chromatography, Bentham open 2016

 https://youtu.be/9MU9sUCI0hg

 https://youtu.be/YXj_Nx5gWK8

 http://www.waters.com/waters/en_US/Chromatography-Consumables-%26-
Columns/nav.htm?cid=505552&alias=Alias_columns_CHEMISTRY
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