High Performance

Liquid
Chromatography
Submitted to: Dr. Mohammad DAKDOUKI 1 Presented by: Fatima Jannoun
 01 Introducti
What is Liquid Chromatography?

on
Introduction to the
02 A r tAnalysis
Dyes i c l e Methods

Development Of A
03 Generic Analysis
M e t hand
Natural o d synthetic dyes analysis by
UHPLC

04 Results and
Performance and Chromatogram
Interpretation
Discussion

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0
1 What is Liquid
Chromatography?
 Liquid Chromatography
Liquid Chromatography is a
Advantages of LC over
chromatographic technique in which we GC
 Separates any compound that is soluble in a
inject the sample in solution form into a
liquid phase.
mobile phase that is liquid.
 Separates biological compounds, natural and
synthetic polymers, and inorganic compounds.
LC is currently the dominate type of  Liquid MP allows LC to be used at lower
chromatography and is even replacing GC in temperatures.
its more traditional applications.  LC better suited than GC for compounds that are
nonvolatile and thermally labile.

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 High Performance Liquid Chromatography
 High Performance Liquid Chromatography (HPLC) is a process of separating components in a
liquid mixture.
 A liquid sample is injected into a stream of solvent (mobile phase) flowing through a column
packed with a separation medium (stationary phase).

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 High Performance Liquid Chromatography

 As bands emerge from the column, flow carries them to one or more detectors which deliver a
voltage response as a function of time. This is called a chromatogram.

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 Basic HPLC System Components
 Solvent Degasser – removes air gases from the
solvents as they flow to the HPLC pump.

Solvent  HPLC pump – provides solvent flow and

Degasser proportioning.

 Autosampler – injects samples into the solvent flow

Detector
provided by the pump.
HPLC
Pump  Detector – produces a signal output for the
software.
Column
Autosampler
 Column Oven – keeps a stable temperature for
reproducible separations.

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 Isocratic vs. Gradient Elution Modes
▧ Isocratic Elution ▧ Gradient Elution
 A single composition of solvents  The composition of solvents is
is used along the separation changed.
 Later eluting peaks are broader  Peaks are sharper throughout
than earlier eluting peaks the chromatogram
because of dispersion  Chromatogram run times may
 Intractable materials build up be shorter when compared to
from sample injections isocratic elution

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 Available Detectors for HPLC
▧ UV/Vis Detectors
 Responds to chromophoric analytes in the range 190 – 800nm

 Single wavelength monitoring

 Good for the majority of organic analytes

 Gradient elution compatible

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 Available Detectors for HPLC
▧ Photodiode Array (PDA) Detectors
 Responds to chromophoric analytes in the range 190 – 800nm

 Multi-wavelength monitoring

 Complete spectral profiles of chromatographic peaks

 Good for the majority of organic analytes

 Gradient elution compatible

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 Available Detectors for HPLC
▧ Fluorescence Detectors ▧ Mass Spectrometric
Detectors
 Responds only to analytes which  The most discriminating detector.
fluoresce naturally.  Delivers mass information for
analyte.
 Extremely sensitive.
 Highly sensitive.
 Gradient elution compatible.  Gradient elution compatible.

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 HPLC Columns
 Traditional HPLC columns are packed with porous silica particles.

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 HPLC vs UHPLC

▧ An advanced LC method, known as ultra-high performance

liquid chromatography (UPLC), has been recently developed.

▧ A high-performance particle stationary phase (particle size from

1.7 to 1.9 μm) and an ultra-high pressure infusion pump are used in
this method.

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HPLC vs UHPLC

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0
2 Introduction to the
Article

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16
 Abstract
Different The development of a
chromatographic time, costs and sample The method was
techniques are used to material saving method applied on a complex
analyze natural and suitable for a wide variety dye mixture containing
synthetic dyes separately, of organic colorants using nearly 130 natural-
since the classes differ ultra-high-performance and synthetic-dye
significantly in chemical liquid chromatography reference compounds.
properties and, therefore, coupled to a photo-diode-
chromatographic array detector.
behavior.

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 Introduction

▧ We set out to ▧ We aim to the ▧ Our objective is to use

develop a novel computer- the optimized method
highly-optimized optimization to create a compound
UHPLC method for strategy (PIOTR) to library based on
the simultaneous optimize a gradient- retention times and
analysis of acidic, elution method for a PDA spectra obtained
basic and neutral large number from the most
dye components in (nearly 130) natural commonly applied
a single and synthetic dye dyestuffs in the field of
experiment.  components. cultural heritage.

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0
3Development Of A Generic
Analysis Method

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 Method
Seven UHPLC columns were tested with different
gradient-elution programs,
Analyze fibers different eluents,
from furniture tapestriesflow
and rates,
Development
Halpine et al used a
water/acetonitrile
Wouters and colleagues gradient with 0.1% Van Bommel et al. A computer-optimization tool called
temperatures, and runfrom
lakeintroduced
pigments times.
a watercolor box 
Based on a reversed-phase mechanism
an LC method trifluoroacetic acid LC × LC
performed LC analyses. method development is complex
‘program for interpretive
using photodiode-array (TFA)  gradient
They applied two Employed gradient
optimization elution using water and
of two-dimensional
The differences in performance between 5% phosphoric and time consuming.
(PDA) detection for red methods.
methanol with 1% formic acid.
resolution’
anthraquinone dyes
acid and 1% FA were also evaluated.
 The second method
The sensitivity of this A novel highly- allowed
method
1985 Later 1996 2013 2017 2016 using tetrabutylammonium as
Later Now optimized UHPLC
determining kermesic acid in insect species often used
an ion-pairing agent method for the
to dye red.
The first was similar to the phosphoric acid method simultaneous analysis of
developed by  The authors replaced FA by
Wouters, except that a different C18
Serrano et al. described the
Pirok et al. applied
acidic, basic and neutral
0.5% phosphoric acid for the Neutral and basic dyestwo-
comprehensive showed better peak shapes
column was used.
analysis of natural dyes, which
performance and method dye components in a
development of a UHPLC
anddimensional
higher peak capacities
LC (LC × LC) to with the phosphoric-acid
could then also be applied to analyze different types of single experiment
system for the method.
purple and blue indigoid dyes synthetic dyes and their
characterization of natural
degradation products in one
dyestuffs
method.

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 Materials and Methods
Chemicals
The mobile phases were prepared using methanol  The flow rate was set at 0.2 ml·min−1
and acetonitrile, FA, NaOH, Dimethyl sulfoxide and 100  The column oven was held at 40 °C.
natural and synthetic reference dyestuffs.
 The chromatographic columns used were a
BEH shield RP C18 column from waters and
and a Zorbax eclipse RRHD C18 column from
Instruments Agilent.

Waters Acquity H-class UHPLC system  Both columns were protected with guard
equipped with a quaternary solvent-delivery columns containing the same c18 packing
system, a column oven, an autosampler and a
PDA detector.

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 Preparation and optimization of mobile phases
 Ultrapure water and methanol or acetonitrile are used

 The eluent was prepared by mixing buffered water and MeOH in ratios of 95/5 for
mobile phase A, and 5/95 for B, respectively. 

 TEA was added as an ion-pairing agent to both A and B at a concentration of 1 or

5 mM, or was left out of mobile phase B entirely.

 In addition to MeOH, analyses were also executed with ACN as the organic modifier
to compare the performance of the two modifiers.

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0
4 Results and
discussion.

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 Chromatographic column
 The first step towards optimization of the method was the comparison of the two types of
stationary phases used by Serrano et al. and Pirok et al. a BEH Shield C18 and a
ZORBAX Eclipse Plus C18 RRHD column, respectively.

 A gradient was applied with mobile phase B increasing from 5% to 95% from 1.50
till 20.0 min, isocratic between 20.0 and 25.0 min, then again from 95% to 5% B
within 2 min, and finally isocratic for 5 more minutes.

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 Chromatographic column
 The chromatogram obtained with the ZORBAX column shows narrower peaks and less
tailing when compared to the BEH Shield column.

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 Chromatographic Columns
 The broader peaks observed for the BEH Shield column may be explained by the additional
hydrophilic carbamate groups of the stationary phase, which increase the affinity of very
acidic compounds.

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 Chromatographic Columns
 Natural dyestuffs, in contrast to synthetic dyestuffs, contain mostly neutral or slightly
acidic compounds, which is why natural dyes show narrower peak shapes on the BEH
column and also why this column has proven to work well for natural dyes before.

The latter, however, shows chromatographic behavior more like that of an acid dye, due to the
presence of two sulfonic acid groups.

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 Chromatographic Columns
 Other compounds that showed poor peak shape on the BEH column contain either one or
more sulfonic-acid groups that is charged at a pH of 3.
 These charged groups may undergo specific interaction with free amide groups on the
surface of the stationary phase of the BEH column, resulting in increased band broadening.
 This effect is not present with the ZORBAX C18 column as the stationary phase does not
contain amide groups and free silanol groups.
 As the stationary phase of the ZORBAX column yielded a more homogenous performance
for this wide set of acidic, neutral and basic compounds, further experiments were conducted
using this column. 

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 Effect of organic modifier and ion pair

 The effect of the composition of the

mobile phase on the chromatographic
performance was studied by varying the
concentration of the ion-pairing agent
between 1 and 5 mM TEA in mobile  In addition, the effects of
phase A and B, and by omitting TEA MeOH or ACN as organic
from eluent B during analyses of
modifiers were studied. 
mixtures A and B.

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 Effect of organic modifier and ion pair
 Increasing the concentration of TEA from 1 to 5 mM resulted in improved peak shapes
and an increased peak capacity.
  Improved peak symmetry was especially observed for compounds that possess multiple
charged sites. 

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 Effect of organic modifier and ion pair
 The decreased retention may be caused by competition between the dye and TEA
for interaction with the stationary phase, or by ion pairing of TEA with remaining
silanol groups on the stationary phase.

 Several compounds that elute later at a higher concentration of TEA are those that
contain additional sulfonate. 

 The improved peak shapes and increased retention times for acid dyes with 5 mM TEA
compared to 1 mM TEA can be assigned to an increased neutralizing effect of the ion-
pairing agent.

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 Effect of organic modifier and ion pair
 When TEA was omitted from mobile phase B, a narrower retention window was observed,
with poor peak shapes, especially for later eluting peaks

 If both mobile phases contain an equal amount of TEA, the ion-pair concentration will remain
constant throughout the analysis.

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 ACN VS MeOH
 For MeOH the retention lines are distributed
 Analyses with ACN resulted in
more evenly than for ACN, which implies that
significantly shorter retention times
many of the compounds show different elution
for all analytes
behavior.

 The lines in these graphs represent all 127  Based on these graphs, and because ACN is
dye components and show their retention less environmental friendly and more
characteristics (ln k) at any organic expensive, further experiments were carried
modifier fraction. out with MeOH.

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ACN VS MeOH

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 Analysis of historical objects
 To assess the performance and applicability of the TEA method for the characterization of
historical artefacts containing natural and synthetic dyes, historical objects were analyzed

 The same samples were also analyzed by the LC × LC method described by Pirok et al.

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Analysis of historical objects

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Analysis of historical objects

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Analysis of historical objects

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 Conclusion
 A UHPLC-PDA analysis method for natural and synthetic dyes was developed and a compound
library for nearly 130 dyestuff compounds was created.

 The TEA method described in this paper was successfully applied for the characterization of
historical artefacts containing synthetic and natural dyes.
 Comparison with the LC × LC method for natural and synthetic dyes proved that this method is
equally capable of analyzing a broad range of acidic, basic and neutral dye components in a
single run, which circumvents the need of having to perform different methods on the same
sample, saving precious sample material and time in the lab.

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 Thanks!
Any questions?

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 Reference

1. Groeneveld, I., Pirok, B. W. J., Molenaar, S. R. A., Schoenmakers, P. J. & van Bommel, M. R.
The development of a generic analysis method for natural and synthetic dyes by ultra-high-pressure
liquid chromatography with photo-diode-array detection and triethylamine as an ion-pairing agent. J.
Chromatogr. A 1673, 463038 (2022).

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