ULTRA

PERFORMANCE
LIQUID
CHROMATOGRAFY
Presented by :
Kaveri Shivaji Aher.

M.Pharm
Quality Assurance Technique

Guided by :
Dr. A.B.Thomas.

Dr.D.Y.Patil Institute of Pharmaceutical

Science and Research Center, Pimpri ,Pune
18.
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UPLC ,Wikipedia
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Introduction
 In 2004 separation science was revolutionized by
‘’Ultra Performance Liquid Chromatography’’ .
 To achieve the dramatic change in 1] Resolution
2] Speed
3] Sensitivity
 Based on

Fig. no. 1
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 Improvement in
UPLC :
 Higher Linear Velocity,
gives lower minima &
less curvature.
 Hold for 1min.
 Rapid injection ,low
volume.
 Auto sampler is
available.
 Higher efficiency
 higher temperature
reduce the viscosity of
mobile phase.
 cooler maintain the
temperature.
 decrease in retention
time
 Increase in Resolution.
Table. no. 1
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Table no. 2 Comparison between HPLC and UPLC

Open Chemistry Journal ,2016, volume3
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Fig. no. 4
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Fig. no. 5
©2012 Waters Corporation
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Principle
 UPLC, HPLC both are based on Van Deemter Equation from 1956.
 Explain correlation between flow rate and plate height.
H = A + B/ν + Cν
Where,
1] H = HETP ( Height equivalent to theoretical plate)
H= L/N
Where,
L = Length of column
N = No of theoretical plate

Fig. no.6
2] A,B,C are Constant.
A = Eddy’s diffusion Fig. no.7

B =Longitudinal diffusion ,at high flow rate ,this effect is smaller

so that is divided by ‘ν’
C =Analyte mass transfer between the pores of stationary phase
and mobile phase.
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Height equivalent to theoretical Plate ( HETP), the smaller the better .

H = L /N

Fig. no.8

Austian Publication Group , Taleuzzaman M.

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Chemistry of Small Particle Size.

 Resolution, speed, efficiency, velocity.
 Resolution equation
Rs =√N/4 (α - 1/α ) ( k/k+1)
where, α=selectivity factor
N = efficiency
k = proportionality constant
Resolution increases with increase in efficiency. And
also increases sensitivity.
N α L/ particle size(dp)
 Efficiency has direct
relationship with Width.
H α 1/W
 N α 1/W²

Fig .no.9
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Contribution of Small Particle Size

 UPLC required Porous Particle.
 SILICA particles : applicable for narrow pH range
 Polymeric particles : no any pH limitations ,low efficiency
 Hybrid Particle : In 2000 ,1st Generation utilize Sol –Gel
synthesis to create durable column that incorporate carbon in
the form of methyl group.
2nd Generation BEH ( Bridged ethylene hybrid)

Efficiency

of L/Particle size
Column

Fig no. 10
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UPLC: A preeminent technique in pharmaceutical analysis Fig. no.11

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 Case Study
of analysis
of 12
phthalates
by HPLC
& UPLC.

Table no. 3
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Fig. no. 12

Chromatographia 2008, 68, November (No. 9/10) Full Short Communication

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Instrumentation

Fig no.13
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Fig. no. 14

© Waters Corporation 2014

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Instrumentation
1.Binary Solvent Manager ( high pressure pump)
- Moves solvent through system .
- Provide steady and ( pulse free) solvent flow.
- Eg :
Solvent High Pressure.
flow rates
a. 1ml /min 103,421kPa
(1034bar,15000
psi)
b. 2ml /min 62.053kPa
(621bar,9000psi
)at reduced
pressure.
Table no.4

 It can pump two solvent simultaneously. Fig. no. 15

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Working :

Consist of 2 serial independent

Pump system.
A B

on left side on right side

L linear drive R linear drive
Pressure Pressure
controller controller
Flow pump flow pump
A B

Combine two solvent

At fliter
flow to the
sample manager
 Capacity to deliver 15000psi
for the optimum flow rate. Fig .no. 16
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2 .Sample Manager (sample injector)

Fig. no.17 a Fig . No.17 b

Challenges : 1) Robust sealing of the needle at higher pressure.
2) Perform pulse free injection process to protect column from extreme
pressure.
 Sample injection can perform approx.15 sec. 1st injection require overtime.
 Sample injection manager maintain samples at any temperature between 4º
to 40º in 25º c (77º F) or less ambient temp. 5º to 9º F.
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3. High Temperature Column Heater / cooler:

 It is identical to column manager.
 Their Dimension of compartment is 1.0mm to 2.1mm & 150mm length.
Features :
 Heating from 5º above ambient to 90ºc
 Consistent temperature control of a single ACQUITY Column.
 Designed to minimize dispersion for UPLC with dead volume.
 Record life time column History .

Fig. no. 18
 4.Columns
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 Column packed with different technology. Table no. 5

Hybrid Particles Silica Based Hybrid Particle

1.7µm 1.7µm 1.8µm

• High retentive for basic compounds. • Good separation for • High retentive for polar
• Excellent peak shape. basic compound organic compounds and
• Good Universal challenge for wide under low pH range. metabolite.
variety of compound. • Fast equilibration • High strength silica particle
• Stable across wide pH range. process. for UPLC separation.
• For separation at high temp. 80 ºC. • CSH Phenyl hexyl • HSSC18, HSST3
• Highest column efficiency. ,CSH C18 ,CSH- fluoro
phenyl
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Fig no.19 BEH particle technology

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Detector
 Detector sampling must be high enough to capture enough data
point across the peak for accuracy.
 Detector cell have minimum dispersion volume to preserve
separation efficiency.
 Detection should 2-3 times higher than HPLC separation.

 Types

1] Evaporative
light scattering
detector :
- incorporate flow
type nebulizer.
- Charged aerosol
detector (CAD)
- less volatile
property than the
mobile phase. Fig. no. 20
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1] Fluorescence detector :
- Multi-channel, multi wavelength fluorescence detector.
- low volume
- high intensity of Hg – Xe lamp in design that minimize
stray light that enhancing the quality of stray light.
- wavelength 200 nm to 800nm excitation rang .

Fig. no. 21 Fluorescence Detector

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Different types of UPLC system

Fig . No.22
H class , H class Bio attribute.
• Based on Acquity UPLC.
• For Biocompatible fluids for biological
molecule.
• True UPLC Performance.

©2012 Waters Corporation7

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I class
 Compatible with an existing Acquity UPLC system .
 Highest point of chromatographic performance.
2D Technology
 2 Dimensional UPLC separation.
 Most challenging assay required this technique.
 Characterize complex sample.
 Illuminate unwanted interferences.
 Mobile phase flexibility. Use multi pump ,multi valve.
 Online sample preparation. Increase sample loading capacity.
 Parallel column regeneration.
 1st sample passes through 1st column and
2nd portion of effluent are send to the 2nd column.
Increase selectivity.
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2D Technology

Fig. no. 23
©2012 Waters Corporation7
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Disadvantages :
 Higher Back Pressure compare to conventional HPLC.
 Decrease the life of column.
 Increase the life of column temperature, reduce the back pressure
problem in UPLC.
 The particle of less than 2micron are mostly non –regenerable. Have a
narrow use.
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Application :
1. Determination of Particle in Groundwater.
2. Rapid Dose Formulation Analysis.
3. Analysis of Traditional Chinese Medicines.
4. Multi –residue Analysis of Pharmaceuticals in Waste Water.
5. Identification of metabolite.
6. In manufacturing / Quality Assurance(Q.A.) /Quality control.(Q.C.)
7. Impurity Profiling.
8. Method Development /Validation.
9. Forced Degradation Studies.
10. Dissolution Testing.
11. Bioequivalence / Bio analysis study.
12. Toxicity Studies.
13. Analysis of Explosives.
14. Analysis of Contamination in Foodstuffs
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Application
16. Determination of Phytoconstituents .

Fig. no.24 Chromatogram of the analysis of Coumarins by HPLC and UPLC

Principle, Instrumentation, and Applications of UPLC Open Chemistry Journal, 2016, Volume 3
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References :

 Swartz ME. Ultra performance liquid chromatography (UPLC): An introduction.

Lc Gc North America. 2005:8-14.Principle, Instrumentation, and Applications of
UPLC: A Novel Technique of Liquid Chromatography

 Chawla G, Ranjan C. Principle, instrumentation, and applications of UPLC: A

novel technique of liquid chromatography. Open Chemistry Journal. 2016 May
6;3(1).

 Swartz ME. UPLC™: an introduction and review. Journal of Liquid

Chromatography & Related Technologies. 2005 Apr 1;28(7-8):1253-63.

 Kumar A, Saini G, Nair A, Sharma R. UPLC: a preeminent technique in

pharmaceutical analysis. Acta Pol Pharm. 2012 May 1;69(3):371-80.

 Waters ACQUITY UPLC Family UPLC Users Meetings Denmark April, 26-27th
Frederic Forini Waters European Headquarters
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References :
 Wu T, Wang C, Wang X, Xiao H, Ma Q, Zhang Q. Comparison of UPLC and
HPLC for analysis of 12 phthalates. Chromatographia. 2008 Nov 1;68(9-10):803-
6.

 Nováková L, Matysová L, Solich P. Advantages of application of UPLC in

pharmaceutical analysis. Talanta. 2006 Jan 15;68(3):908-18.

 Richard JP. Time-delay systems: an overview of some recent advances and open
problems. automatica. 2003 Oct 1;39(10):1667-94.

 Taleuzzaman M, Ali S, Gilani SJ, Imam SS, Hafeez A. Ultra performance liquid
chromatography (UPLC)–a review. Austin J Anal Pharm Chem. 2015;2(6):1056.

 www.google.com
 Thank You.

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