Bachelor Thesis Scheikunde
Characterization of Synthetic dyes by
Comprehensive Two-Dimensional Liquid
Chromatography
door
Jitske Knip
30 juni 2015
Studentnummer
10367152
Onderzoeksinstituut
Universiteit van Amsterdam
Onderzoeksgroep
Analytische Chemie
Verantwoordelijk docent
Dhr. prof. dr. ir. P.J. (Peter)
Schoenmakers
Begeleider
Dhr. B.W.J ( Bob) Pirok Msc
Analyse van Synthetische kleurstoffen met behulp van 2-dimensionale
vloeistofchromatografie
Ongeveer halverwege de negentiende eeuw was een wetenschapper genaamd William H. Perkin, bezig met de
ontwikkeling van een medicijn tegen malaria. Terwijl hij hard aan het werk was op het lab, maakte hij per
ongeluk in plaats van werkend medicijn een felle paarse poeder. Deze mooi gekleurde poeder is uiteindelijk
geregistreerd als de eerste echte synthetisch gemaakte kleurstof. Perkins product was zo populair dat hij en
zijn medewetenschappers zich richtten op de ontwikkeling van meer van dit soort kleurstoffen, en tegen het
eind van de negentiende eeuw waren er al meer dan vierhonderd kleuren gemaakt en geregistreerd. Het
succes van deze uit het laboratorium afkomstige kleurstoffen zorgde ervoor dat zij al gauw de natuurlijke
kleurstoffen, die al sinds de prehistorie gemaakt en gebruikt werden, hadden vervangen. Hun succes was te
danken aan het minder arbeidsintensieve en langdurige productieproces en hun desondanks fellere kleuren.
Maar waarom is dit interessant voor wetenschappers? Waarom zou een scheikundige zich bezig houden met
de analyse van deze stofjes. Om te beginnen is er veel interesse naar het productieproces van die tijd, de
negentiende eeuw is immers al ruim honderd jaar geleden. Maar nog belangrijker is de interesse in het
degradatie proces die deze kleurstofjes over de loop van tijd ondergaan. Blootstelling aan UV-licht voor
misschien wel meer dan honderd jaar, zorgt ervoor dat een kleurstof gaat vervagen. Door te analyseren hoe
dit gebeurd, is het misschien mogelijk om dit degradatie proces te vertragen, of om op z’n minst te kunnen
achterhalen hoe het er origineel uit heeft gezien. Daarom is dit project opgezet. Het doel van dit project was
om een scheidingsmethode te ontwikkelen die de verschillende soorten kleurstoffen (zuur en basisch) kan
tegelijkertijd kan scheiden. Dit wordt gedaan met behulp van vloeistofchromatografie. Eerder onderzoek heeft
al aangetoond dat het moeilijk is om zure en basische kleurstoffen tegelijkertijd te kunnen scheiden, hierom
wordt er tijdens dit onderzoek gebruik gemaakt van 2-dimensionale vloeistofchromatografie. Dit betekent dat
we de scheiding gaan laten plaatsvinden op basis van 2 verschillende scheidingsmechanismes, maar dan wel
tegelijkertijd! Deze methode wordt niet alleen gebruikt omdat het moeilijk is om deze twee soorten
kleurstoffen van elkaar te scheiden, maar ook omdat de hoeveelheid synthetische kleurstoffen zo groot is, dat
het onmogelijk lijkt om ze allemaal van elkaar te kunnen scheiden op slecht één chromatogram. Een
chromatogram bestaat namelijk uit pieken, die elk een representatief zijn voor een unieke stof. Er is echter
maar gelimiteerd ruimte op zo’n chromatogram om deze pieken weer te geven en het chromatogram van een
één dimensionale methode heeft gewoon niet genoeg ruimte om de hoeveelheid verschillende pieken die dit
onderzoek omvat weer te kunnen geven. Een 2-dimensionaal chromatogram biedt echter wel genoeg ruimte
voor een dergelijk grootschalig onderzoek.
Daarom is tijdens dit project gewijd aan het
ontwikkelen van een methode waarmee een
mix van welgeteld 54 verschillende
synthetische kleurstoffen van elkaar
gescheiden kunnen worden, wat uiteindelijk
is gelukt. Zelfs scheiding van de zure en
basische kleurstofjes is hiermee bereikt. Dit
is
bewerkstelligd
door
twee
scheidingsmethodes (Reversed Phase en Ion
Exchange Chromatography) met elkaar te
combineren en zo een 2-dimensionaal
chromatogram te creëren. Dit is jammer
genoeg slechts de eerste stap geweest naar
Voorbeeld van een chromatogram van een 2-dimensionale
het uitvoeren van degradatie studies, welke
methode (IEC × RP)
juist zo belangrijk zijn.
2
Characterization of Synthetic Dyes by
Comprehensive
Two-Dimensional
Liquid Chromatography
Abstract:
The analysis of early synthetic dyes is interesting for several reasons. But most importantly, it can
help with the identification of possible degradation products that have formed over time. The
analysis of these degradation products can help with the clarification of the original appearance
and/or the composition of these dyestuffs. To analyse these dyestuffs UHPLC can be used. Because
of the great sample amount and the difficult separation of acidic and basic dyes simultaneously,
comprehensive 2-dimensional LC was applied to increase peak capacity and induce better
separation. To separate the dyestuffs based on hydrophobicity and charge, reversed phase ion pair
chromatography and strong anion exchange chromatography were used. The application of these
two separations methods in a comprehensive IEC×RP setup led to the separation of 54 different
dyestuff samples. As well as the simultaneous separation of acidic and basic dyes.
1
Table of Contents
Analyse van Synthetische kleurstoffen met behulp van 2-dimensionale vloeistofchromatografie ... 2
1. Introduction .................................................................................................................................... 3
1.1 Dyestuffs ................................................................................................................................... 3
1.2 Liquid Chromatography ............................................................................................................ 4
1.3 Application of Comprehensive Two-Dimensional Liquid Chromatography to Analysis as
Dyestuffs ......................................................................................................................................... 4
1.4 Retention Mechanisms ............................................................................................................. 5
2. Experimental ................................................................................................................................... 8
2.1 Equipment ................................................................................................................................. 8
2.2 Ion pair chromatography .......................................................................................................... 8
2.3 Strong Anion Exchange Chromatography ................................................................................. 9
2.4 Two Dimensional method ......................................................................................................... 9
2.5 Sample Preparation................................................................................................................. 10
3. Results and Discussion .................................................................................................................. 11
3.1 Ion pair chromatography ........................................................................................................ 11
3.2 Strong Anion Exchange Chromatography ............................................................................... 12
3.3 Van Deemter ........................................................................................................................... 14
3.4 Comprehensive 2D-chromatography ...................................................................................... 15
3.5 Hydrophilic Interaction Chromatography ............................................................................... 19
4. Conclusion and Perspectives......................................................................................................... 20
5. References .................................................................................................................................... 21
2
1. Introduction
1.1 Dyestuffs
When William H. Perkin was trying to synthesize an anti-malarial drug, quinine, in 1856, he
accidentally discovered a brilliant purple dye called Mauve, what is now considered the first ‘real’
synthetic dye.[1] The synthetic dye was such a great commercial success that within a few years,
scientists developed even more synthetic dyes and by the end of the century, in 1897, already 404
different dyestuffs had been synthesized. Even though natural colorants had been used since the
prehistoric times, the huge success of the synthetic dyes led to their disappearance, as they were
quickly replaced by the synthetic ones.[2]
The analysis of these synthetic dyes can be very useful for various reasons. For example, it is
helpful to understand not only the production process, but also to explain the degradation processes
or even to figure out how to slow down the rate of decay of objects of art. To understand more
about the original appearance and obtain even more
knowledge of the historical context of particular objects, an
option is to identify the dyestuffs that were used for the dying
of textile.[3] It may also be interesting and useful to identify
the degradation products that might have formed over time
whilst the dyestuff decay. The identification of these
degradation products can help with the clarification of the
original appearance and composition of the dyestuffs back
when they were first made and used, maybe several hundreds
of years ago. One way to analyse these early synthetic
dyestuffs and their degradation products is by using ultra
high-performance liquid chromatography (UPLC), a technique
that enables rapid analysis and quick identifications of the
different classes of dyestuffs.[2]
In general, the early synthetic dyestuffs can be divided into
different categories. There are acid, basic, mordant and direct
dyes. This division is based on the binding mechanism that
takes place when the dyestuff binds to textile fibers. Of these
four, the most important and frequent class is the acid dye
class. This class of early synthetic dyes derive their name from
the fact that they are applied to textile fibers in an acid dye
bath. The functional group that is responsible for the bond
with the textile fiber that forms during this dying process is
the acidic group(s) they possess, such as a sulfonic acid and/or
a nitroso group. Thus basic dyes, as one would suspect, are
applied using basic solutions. The groups responsible for these
basic characteristics are one or more primary or secondary
amine groups. Direct dyes are used in a neutral environment
and bind directly to the textile fiber, this bond is caused by Figure 1: Examples of a Direct, acid,
hydrophobic interactions. Even though functional groups may basic and mordant dye
3
be present in the structure of the dyes of this group, they do not take part in the binding process to
the fiber. The most complex class is that of the mordant dyes, where a transition metal, like
chromium, aluminum, tin or iron, are responsible for the bond between dye and fiber.[16] Examples
of these different categories of dyes can be seen in Figure 1.
1.2 Liquid Chromatography
Ultra high-performance liquid chromatography is the more improved form of the standard Highperformance Liquid Chromatography (HPLC). It works along the same principles, except where HPLC
is limited to pressures of 400 bar, UPLC can go up to 1200 bar. This enables the use of columns
packed with smaller particles and as a result shorter run times are obtained. In liquid
chromatography, a mobile phase is utilized, in the form of a flowing liquid, and a stationary phase is
present, which consists of sorbents packed inside a column.[4] The combination of these two phases
will induce the separation of the sample components as a result of differences in partitioning
between the two phases. These interactions will cause them to each have a different degree of
retention and leave the column at varying times.
There are different types of HPLC techniques, that involve different stationary and mobile phases.
Some of the most commonly used techniques are: Reversed Phase Chromatography (RPLC), Normal
Phase Chromatography (NPLC), Ion-Exchange Chromatography (IEC), Hydrophilic Interaction
Chromatography (HILIC) and Size-Exclusion Chromatography (SEC). Each of these techniques rely on
a different type of interaction, e.g. with NPLC where the sample components are separated on basis
of their affinity for a polar stationary phase.[5] But not just molecular properties like polarity can be a
determining factor in liquid chromatography. When using the SEC mode, the size is all that matters.
In size-exclusion chromatography, separation arises from the relative size or hydrodynamic volume
of macromolecules.[6] Consequently, the sample is separated on size. Both of these techniques are
very useful when trying to separate very polar components or macromolecules respectively, but
seeing that the dyestuffs analysed during this research are neither, we neglect these two techniques
and focus on the remaining ones, all of which will be explained extensively later on.
1.3 Application of Comprehensive Two-Dimensional Liquid Chromatography to Analysis as Dyestuffs
1.3.1
Why
use
Two-Dimensional
Liquid
Chromatrography?
Even though there is a lot of knowledge and
information on the early synthetic dyestuffs,
analyzing them can be difficult. Firstly because
most of these early dyestuffs that were created in
the 1900s were not created to be perfectly pure to
begin with, as long as they worked well enough,
they were good enough. On top of that there is also
the possibility that the dyes will have decayed
somewhat over time and this leaves us with one or Figure 2: Example of a contour plot (2D)
more degradation products, making these samples
4
hard to separate and identify. Also considering the limited peak capacity of 1D chromatography, and
the enormous amount of synthetic dyes that have to be analysed, it was decided that onedimensional chromatography would not offer sufficient separation power. Previous research of onedimensional chromatography with these type of samples furthermore showed that it was difficult to
separate acidic and basic dyes simultaneously. Therefore comprehensive two-dimensional
chromatography (LC×LC) was introduced, to separate these compounds and degradation products
on a single chromatogram. Not only to allow for greater peak capacities, but also to be able to
separate the acidic and basic dyes simultaneously2]
With the fundamentals of liquid chromatography explained, this section will address the
application of the techniques for the analysis of several dyestuffs. It will explain how the
combination of two different LC methods will allow for even greater separation and result in a
comprehensive two-dimensional chromatogram. While two-dimensional liquid chromatography
often has been applied by simply taking interesting cuts (heartcut) from a first dimension and
running these through a second dimension, the entire first dimension eluent is subsequently
subjected to the second separation dimension in comprehensive two-dimensional chromatography
(LC×LC).
Two-Dimensional liquid chromatography is a technique that makes use of two different types of
subsequent columns. The columns are placed in series and the analytes will be run through both of
them subsequently, having the analytes retain using two varying factors, for example hydrophobicity
and size. This will result in a chromatogram with two separate dimensions. An example of a contour
plot (2D) can be seen in figure 2.[9] For the dyestuffs, reversed phase and anion exchange
chromatography were chosen to use for the two dimensional analysis. These retention mechanisms
were selected due to the hydrophobic backbone and the varying charges that the dyestuffs display.
1.3.2 Orthogonality and eluent compatibility
When performing two dimensional chromatography there are certain aspects that have to be
considered. One of those aspects is the orthogonality of the two different separations, where the
separation arising from the two separation dimensions is needs to be as statistically independent as
possible from one another. Indeed, it is not useful to separate the samples twice based on the same
separation principle. And on the other hand the eluent compatibility between the two dimensions is
something that needs to be considered, as it can create restrictions on the possible 2-D
combinations. This is because the eluent from the first dimension is principally the sample solvent in
the second dimension and will not always be compatible with this second dimension, for example,
when your second dimension is normal phase and the mobile phase of the first dimension consist of
mainly water, the normal phase separation will not be successful, as the samples will elute with the
water and have little to no retention.[11]
1.4 Retention Mechanisms
1.4.1 Anion-Exchange Chromatography
Unlike reversed-phase and normal-phase chromatography, analytes are not separated based on
hydrophobicity in ion-exchange chromatography. With ion-exchange chromatography the
separation is based on differences in electrostatic interactions. It is designed for the separation of
differently charged or ionisable molecules. In ion-exchange chromatography, the mobile phase
typically is an aqueous buffer and the stationary phase is an inert organic matrix covered with
5
ionisable functional groups that carry an exchangeable oppositely charged counter-ion (either cation
or anion).[8] If the analyte contains a charge, opposite to that of the organic matrix, it will displace
the counter-ion and adsorption of the analyte to the matrix takes place. There are two types of ion
exchange chromatography: weak and strong. When executing weak ion exchange chromatography
the samples are pH dependent and will elute when the pH is changed in such a way that they no
longer remain charged and desorption takes place, also resulting in the elution of the analyte. Strong
ion exchange, on the other hand, depends on the increase of a similarly charged species within the
mobile phase. This species will then compete with and eventually displace the analyte bound to the
matrix surface. By gradually increasing the salt concentration of the mobile phase, the affinity of
interaction between these salt ions will exceed the interaction of the analyte charges, resulting in
their displacement and thus their elution. The separation arises due to differences in the strength of
the bond between the stationary phase and the varying samples. Should a compound have more
than one oppositely charged group, it will bind more strongly to the matrix than when a compound
is singularly charged, thus they need higher concentrations of competing salts to be displaced.
The LC technique that was selected for the first dimension separation consist of strong anion
exchange. As most of the selected dyestuffs carry varying charges, some of them having negative
charges ranging from 1- to 3-, therefor strong anion exchange chromatography appeared to be a
good choice for the first part of the separation..
1.4.2 Reversed Phase Chromatography with Ion Pair
Reversed-Phase, Ion-exchange and Hydrophilic Interaction Chromatography are all unique
techniques that separate components on different aspects. RPLC is one of the most popular
techniques this is because most relevant analyte mixtures comprise of molecules with differences in
hydrophobicity. RPLC consists of a relatively polar mobile phase and a hydrophobic (nonpolar)
stationary phase. Modern day reverse phase stationary phases mostly consist of permanently
bonded hydrophobic groups, e.g. octadecyl (C-18) bonded groups, on a silica support. The separation
of this technique is attributed to the solvophobic or hydrophobic interactions that take place.[4][7]
The separation technique that was chosen for the second dimension, is Reversed Phase
Chromatography (RPLC). This is because the selected dyestuffs mostly consist of a carbon backbone,
build up from aromatic rings and several functional groups. Thus dyestuffs are organic hydrophobic
compounds, which will have good interaction with the hydrophobic stationary phase of the Reversed
Phase Column (in this case the organic C-18 chains inside the column). This interaction will cause the
retention needed to have a good separation of the analytes.[7]
RPLC is a technique that is very dependent on the affinity between the analytes and the organic
stationary phase and solvent, but also their affinity with the water. When studying the functional
groups that the dyestuffs contain, one can see several charged group. Charges like this will cause
extreme polarity, decreasing their hydrophobicity, which will result in loss of retention. It is possible
to prevent this from happening by using a so called ‘ion pair’ and thus performing Ion Pair
chromatography (ICP). Because when adding an oppositely charged ion to the mobile phase, it will
pair up with the negatively charged functional groups of the analytes, neutralising them entirely.
This will cause them to retain during the reversed phase chromatography like neutrally charged
compounds would, the ion pair can even add to the retention depending on its qualities. Should it
have high reversed phase properties (like long carbon tails), it will enhance the reverse phase
retention and therefor retain the compounds it has paired with even more than it normally would
have.[10]
6
But for the execution of IEC×RP, a cation was chosen with low reversed phase interaction, this
was done because it is important to take orthogonality into consideration. When taking into account
that each negative charge a compound contains will pair up with a cation of the ion pair, the more
negative groups they have, the more they will pair up with the ion pair. This ion pairing will result in
increased retention per pair, making them separate not only on the reversed phase properties they
have, but also based on the amount of negative charges the analyte carries. For regular RPLC this
would not be a problem, often even a desired effect, but for the orthogonality of a comprehensive
2D IEC×RP method, it’s necessary to keep the separation on basis of charge to a minimum during the
second dimension, as it is the main basis of the separation of the first ion exchange dimension.
1.4.3 Hydrophilic Interaction Chromatography
Hydrophilic Interaction Chromatography is a chromatographic technique that is a variant of NPLC, where the retention of the analytes is caused by partitioning between analyte and a hydrophilic
stationary phase and as mobile phase a relatively hydrophobic eluent, usually something like 5-40%
water in ACN. HILIC is used to separate small polar compounds. The retention increases as the
polarity of the mobile phase decreases, this results in greater retention for the more polar
compounds (which is the opposite of RP-LC). [17][12]
7
2. Experimental
2.1 Equipment
For the method development and analysis, an Agilent 1290 Infinity 2D-LC setup was used. The
system utilized two Agilent 1290 Infinity Binary Pumps (G4220A), each equipped with a Jet Weaver
Mixer 380 µL (G4220-60012), two Agilent 1290 Infinity Thermostatted Column Compartments
(G1316C) for both columns, of which the compartment for the second dimension column was
equipped with an Agilent 2-position 8-port valve (G4236A), a schematic representation of this valve
can be seen in figure 3. The system also featured an Agilent 1290 Infinity Autosampler (G4226A) and
two Agilent 1290 Infinity Diode Array Detectors (G4212A) with Agilent Max-Light Cartridge Cell 60
mm (G4212-60007) and Agilent Max-Light Cartridge Cell (10 mm, V(o) 1.0ul) (G4212-60008) flow
cells for the first and second dimension respectively. An Agilent 1290 Infinity In-Line Filter (G50674638) was located directly preceding the first dimension column.
The system was controlled by a computer with Agilent OpenLAB CDS Chemstation Edition (Rev.
C.01.04 [35]). Data was processed and analysed using MATLAB 2013a.
Figure 3: Visual representation
loop/valve configuration
of
the
2.2 Ion pair chromatography
For Ion Pair Chromatography (IPC) the following method was applied. The mobile phase consists
of a gradient of [A]: Water/Acetonitrile (95:5) with 10 mM tetramethyl ammonium hydroxide (TMA),
brought to pH 3.0 with formic acid. And [B]: Acetonitrile/Buffer[A] (95:5). The mobile phase was
delivered at a flow rate of 1.000 mL/min. The gradient profile can be seen in table x. Separation was
performed on a C-18 column (Agilent ZORBAX Eclipse Plus RRHT 50x4.6mm, 1.8 µL)
The column was placed in a column oven and was kept at a constant temperature of 25 ⁰C.
The method above was performed in the same manner with tetrabutyl ammonium hydroxide
(TBA) instead of TMA.
8
Table 1: HPLC gradient for Ion Pair Chromatography, all changes are linear
Time (min)
0
0.50
12.50
13.00
15.00
%A: Water/
Acetonitrile with 10
mM TMA (95:5, v:v)
pH 3.0
100
100
0
0
100
%B:
Acetonitrile/Buffer[A]
(95:5, v:v)
0
0
100
100
0
2.3 Strong Anion Exchange Chromatography
For Strong Anion Exchange analysis the following method was applied. The mobile phase consists
of a gradient of [A]: water/acetonitrile (1:1) and [B]: water/acetonitrile (1:1) with 100 mM
ammonium sulphate. The mobile phase was delivered at a flow rate of 0.500 mL/min. The gradient
profile can be seen in table 2. Separation was performed on a SAX (strong anion exchange) column
(150 mm x 5 mm, 2.1 µm I.D.).
The column was placed in a column oven and was kept at a constant temperature of 25 ⁰C.
Table 2: HPLC gradient for Strong Anion Exchange, all changes are linear
Time (min)
%A: water/
acetonitrile (1:1)
0
0.50
10.50
14.50
15.00
16.00
100
100
0
0
100
100
%B: 100 mM (NH4)2SO4
in
water/acetonitrile
(1:1)
0
0
100
100
0
0
2.4 Two Dimensional method
For two dimensional HPLC two different systems were evaluated and combined. The mobile
phase of the first system consists of a gradient of [A]: water/acetonitrile (1:1) and [B]: a buffer
solution of water/acetonitrile (1:1) with 100 mM ammonium sulphate. The mobile phase was
delivered at a flow rate of 0.010 mL/min. The gradient profile can be seen in table 3. For this system,
separation was performed on a SAX (strong anion exchange) column (150 mm x 5 mm, 2.1 µm I.D.)
The second system has a mobile phase that consists of a gradient of [A] a buffer solution of 10
mM tetramethylammonium hydroxide (TMA) in water/acetonitrile (95:5) that was brought to pH 3
with formic acid, and [B] an acetonitrile/buffer (95:5). The mobile phase was delivered at a flow rate
of 2.500 mL/min. The gradient profile can be seen in table 4. For this system, separation was
performed on a C-18 column (Agilent ZORBAX Eclipse Plus RRHT 50x4.6mm, 1.8 µL)
9
For the performance of comprehensive two dimensional HPLC the systems are linked with an 8port valve, containing two 40 µL loops.
Both columns were placed in a column oven and were kept at a constant temperature of 25 ⁰C.
Table 3: HPLC gradient for first dimension (IEC) system, all changes are linear
Time (min)
%A: water/
acetonitrile (1:1)
0
150.00
170.00
224.00
254.00
264.00
314.00
100
45
20
0
0
100
100
%B: 100 mM (NH4)2SO4
in
water/acetonitrile
(1:1)
0
55
80
100
100
0
0
Table 4: HPLC gradient for second dimension (RP) system, all changes are linear
Time (min)
0
2.2
2.4
2.6
3.0
%A: Water/
Acetonitrile with 10
mM TMA (95:5, v:v)
pH 3.0
100
0
0
100
100
%B:
Acetonitrile/Buffer[A]
(95:5, v:v)
0
100
100
0
0
2.5 Sample Preparation
Standards of the 54 dyestuffs were analysed and are listed in Table b. In this table the colour
index names and numbers are listed, along with other relevant information[1].
For HPLC analysis, the pure dyestuffs were dissolved in water:methanol (1:1) at a concentration
of 5000 ppm. Prior to injection, they were diluted in water:methanol (1:1) to a concentration of 250
ppm. When a sample was still an unclear solution (one could not see through it), it was diluted
further to a concentration of 25 ppm. Of the 54 selected dyestuffs a mixture was made according to
the ratios represented in Appendix 1. Of the separate samples, a volume of 2.5 µL was injected, of
the mixture a volume of 20 µL was injected.
10
3. Results and Discussion
3.1 Ion pair chromatography
When performing reversed phase chromatography with charged samples, using an ion pair can
influence the results of the chromatography significantly. This is called Ion Pair chromatography
(IPC). For the purpose of demonstrating this, a comparison of a chromatogram with and a
chromatogram without the ion tetramethyl ammonium (TMA) can be seen in Figure 4. The
chromatogram shows that when an ion pair is present, the separation is significantly improved and
results in a higher peak capacity. As explained earlier, this is the result of the neutralising effect that
the ion pair of choice (TMA) exhibits.
Figure 4: Overlay of UV chromatograms of the separation of a mixture of 54 synthetic dye
samples by ion-pair chromatography with 5 mM TBA (orange) and TMA (blue) in the mobile
phase at pH 3.0. Gradient analysis . Mobile phase A: buffer, B: acetonitrile. Chromatogram
recorded at 254 nm. Flow: 1.0 mL/min, Column: Agilent ZORBAX Eclipse Plus RRHT 50x4.6mm,
1.8 µm. Injection volume: 1.0 µL.
While the effect of the presence and absence of an ion pair such as TMA is quite evident,
changing the type of ion pair is also of a great influence on the eventual retention. To register the
effects that different ion pairing can
have
on
reversed
phase
chromatography, another experiment
was done to demonstrate these
effects. For this, the exact same
measurements were performed with
the ions tetramethylamine (TMA) and
tetrabutylamine (TBA) in the mobile
phase, both the molecular structure of
Figure 5: Molecular structure of tetrabutylammonium and
tetramethylammonium
11
TMA and TBA can be seen in figure 5. As can be seen in this figure, TBA’s four carbon tails are much
longer than those of TMA. These long butyl groups will increase the hydrophobicity and thus cause
extra reversed phase retention. More than the methyl groups of TMA will. But a large molecule like
TBA will also shield off the analyte is has paired with, making the eventual retention less about the
compounds own reversed phase interaction and more about the ion pairing that takes place at each
negatively charged site. The example of this can be seen in figure 6. When the compounds are paired
with TBA, the retention for the paired compounds is significantly bigger than when TMA is used. As
can be seen in this chromatogram, the greater part of the compounds elute later on in the
chromatogram. The most hydrophobic compounds can be found here, but when using TBA all of the
charged compound also elute around this time. While when using TMA, the compounds elute more
evenly distributed, leaving us with a greater separation and peak capacity.
Figure 6: Overlay of UV chromatograms of the separation of a mixture of 54 synthetic dye
samples by ion-pair chromatography with 10 mM TMA (blue) and no ion pair (red) in the mobile
phase at pH 3.0. Gradient analysis . Mobile phase A: buffer, B: acetonitrile. Chromatogram
recorded at 500 nm. Flow: 1.0 mL/min, Column: Agilent ZORBAX Eclipse Plus RRHT 50x4.6mm,
1.8 µm. Injection volume: 1.0 µL.
3.2 Strong Anion Exchange Chromatography
The first dimension of the comprehensive 2-dimensional LC×LC mechanism is strong anion
exchange (SAX) chromatography, as mentioned earlier, this type of HPLC will allow for the
separation of the dyestuffs that are carrying one or more negative charges. The ones that contain
more negative charges will need higher concentrations of exchanging ions to elute. They stay punt
until the gradient of the salt concentration has increased. Therefore they will be retained longer
than those with only one or no charge (or positive charge). An example of this can be found in figure
7. The compounds that have no charge or those that are positively charged, exhibit little to no
retention by the SAX column whatsoever. They will elute quickly and therefore appear at the
12
beginning of the chromatogram. The charged analytes however, are retained by their negative
charges and thus more to the middle and the back of the chromatogram.
As some of the dyestuffs have multiple negative charges (like those with a charge of 3-), a very
strong buffer anion was required to compete and exchange with them. Therefore ammonium
sulphate was chosen as the anion exchange ion, as it turned out that SO42- is an anion that is suitable
to compete and exchange with these highly negative dyestuffs. Unlike the anion acetate, used
regularly with anion exchange chromatography, that was tried previous to sulphate. But it was not
strong enough to compete with the triple charged samples.
+1/0
-1
-2
-3
Figure 7: Overlay of UV chromatograms of the separation of a mixture of 54 synthetic dye samples
by strong anion-exchange chromatography with ammonium sulfate in the mobile phase. Gradient
analysis. Mobile phase A: water/acetonitrile (1:1), B: 100 mM ammonium sulfate in
water/acetonitrile (1:1). Chromatogram recorded at 254 nm. Flow: 0.5 mL/min, Column: Agilent PLSAX 150x2.1mm, 8 µm. Injection volume: 1.0 µL.
13
3.3 Van Deemter
To limit the impact of the flow rate on the band broadening of the analyte bands, it is important
to determine the optimal flow rate by using the van Deemter equation. This is an equation that
predicts the optimum velocity at which the peak broadening is at a minimum. Peak broadening
happens due to several properties of a separation. The van Deemter equation can be seen below.
𝐻 =𝐴+
𝐵
+𝐶∙𝑢
𝑢
Here, 𝐻 represents the height of a theoretical plate (HETP), 𝐴 the eddy diffusion (m), 𝐵 the
longitudinal diffusion coefficient (m2 s-1), 𝐶 the mass transfer resistance coefficient and 𝑢 the linear
velocity in m s-1.
With this equation it is possible to determine the lowest possible plate height H. The lower H, the
more narrow the peaks. The 𝐴 in this equation stands for the eddy-diffusion. This is the diffusion
that is caused by irregularities in the packing of the column. Because of these irregularities in the
packing the different pathways an analyte particle can take will have varying pathlengths, which will
cause varying retention times for particles of the same analyte, which leads to peak broadening.
Would the column have been ideally packed, all of the possible pathways would have been equally
long and there would be no eddy-diffusion and thus no peak broadening. 𝐵, on the other hand, is
the diffusion-coëfficient and is the parameter that includes the dispersion throughout the mobile
phase. If a measurement is to take a very long time, the dispersion will increase more and more.
Therefore it is important to have a velocity that allows for minimal dispersion. Finally, there is the C
parameter, which represents the contribution as a result of the resistance to mass transfer. The
exact description of the C parameter is rather complex and outside the scope of this study. In short,
the C term accounts for the fact that the equilibrium of analytes between the stationary phase and
mobile phase is constantly disturbed as the mobile phase is constantly moving.
By using this equation it was determined that for the reversed phase column (the second
dimension of the 2D-LC), at all higher flow rates, the theoretical plates arrived at a minimum.
Therefor the maximal flow rate that the column could take was used for the second dimension
(2.500 mL/min).
14
3.4 Comprehensive 2D-chromatography
By applying the two mechanisms discussed above, RP-IPC and SAX chromatography, twodimensional chromatography was executed.
The first 2D method that was applied resulted in the chromatogram shown in figure 8. This
chromatogram was made with a method that contained 5 mM of TMA in the reversed phase mobile
phase and 10 µL of the mixture of 54 synthetic dyes was injected. Also the gradient for the first
dimension was longer than that of the eventual method. This longer gradient can be seen in Table 5.
The result of this is a chromatogram with decent separation and peak capacity.
Table 5: Initial gradient for the first dimension of the 2D-LC measurements
%A: water/
acetonitrile (1:1)
0
270.00
300.00
310.00
360.00
100
0
0
100
100
Sec o nd D im ensio n reten tio n (s) - I on -P a ir R eversed -Ph a se C18
Time (min)
%B: 100 mM (NH4)2SO4
in
water/acetonitrile
(1:1)
0
100
100
0
0
175
150
125
100
75
50
25
0
0
25
50
75
100
125
150
175
200
225
250
275
300
325
350
First Dimension retention (min) - Strong Anion-Exchange
Figure 8: Fig. 39 - LCxLC-UV chromatogram of a mixture of 54 synthetic dyes at 254 nm. Run 0005. Injection
volume: 10 µL. First dimension: [B.026] Agilent PL-SAX 150x2.1mm, 8 µm, flow: 0.01 mL/min. Second
dimension: [B.014] Agilent ZORBAX RRHT Eclipse Plus 50x4.6mm, 1.8 µm, flow: 2.4 mL/min. Measured on
Agilent 1290 Infinity 2D-LC. Ammonium sulfate was used as buffer displacement ions in the first dimension
Figure
9 shows
the0result
the increase
of the
injection volume
from
uL toas20
uL. in the mobile
in a gradient
from
to 100ofmM.
Tetra methyl
ammonium
hydroxide
was10used
ion-pair
phase buffer of the the second dimension at a concentration of 5 mM.
15
Sec o nd D im ensio n reten tio n (s) - I on -P a ir R eversed -Ph a se C18
However, to establish greater visibility of the smaller peaks a greater sample volume (20 µL) was
injected. This modification resulted in an increase of intensity of the peaks, as can be seen in Figure
9.
175
150
125
100
75
50
25
0
0
25
50
75
100
125
150
175
200
225
250
275
300
325
350
First Dimension retention (min) - Strong Anion-Exchange
Figure 9: LC×LC-UV chromatogram of a mixture of 54 synthetic dyes at 254 nm. Injection volume: 20 µL. First
dimension: [B.026] PL-SAX 150x2.1mm, 8 µm, flow: 0.01 mL/min. Second dimension: [B.014] Agilent
ZORBAX RRHT Eclipse Plus 50x4.6mm, 1.8 µm, flow: 2.4 mL/min. Measured on Agilent 1290 Infinity 2D-LC.
Ammonium sulfate was used as buffer displacement ions in the first dimension in a gradient from 0 to 100
mM. Tetra methyl ammonium hydroxide was used as ion-pair in the mobile phase buffer of the second
dimension at a concentration of 5 mM.
The following step of the optimisation process was to decrease the amount of unused space in
the chromatogram. This was done by introducing quick gradients. Therefore a new gradient method
was introduced for the first dimension, which reduced its run time by 44 minutes. The modified
gradient can be seen in Table 3. By applying this gradient the following chromatogram was
established (Figure 10).
16
Effective Gradient
0
50
100
150
H2O/ACN (1:1)
100 mM Ammonium Sulphate in
H2O/ACN (1:1)
200
250
300
Time (s)
100
0
% solvent
Figure 10: LC×LC-UV chromatogram of the separation of a mixture of 54 synthetic dye with effective
gradients. Dual gradient analysis. First dimension: Strong Anion-Exchange, Agilent PL-SAX 150x2.1nm, 8
µm, 0.01 mL/min. Second Dimension: Ion-Pair Chromatography, Agilent ZORBAX Eclipse Plus RRHT
50x4.6mm, 1.8 µm, 2.4 mL/min. Chromatogram recorded at 254 nm. Injection volume: 20.0 µL. Loopsize:
40 µL.
When looking at the chromatogram displayed in Figure 10, one sees that in the middle region of
the chromatogram the peaks are blotched. It seems like they are tailing in the second dimension, the
reversed phase dimension. To decrease this tailing, that seemed to happen for just the negatively
charged analytes, the Ion Pair concentration in the mobile phase was increased to ensure better ion
pairing with the negatively charged compound. Thus the concentration was increased from 5 mM to
10 mM of TMA.
By using 10 mM TMA in the mobile phase of the reversed phase of the second dimension the
following chromatogram was achieved (Figure 11). This chromatogram displays slightly sharper
peaks in the middle regions and high peak capacity in the earlier regions of the first dimension (40 to
80 minutes) and throughout all of the second dimension. As the measurement progresses, the peaks
lose their sharpness and become more stretched out, which is due to the common qualities of ion
exchange column, that rarely produces sharp peaks for strongly charged components. The figure
also displays the effective gradients, which is the method gradient corrected with the dwell time.
The downside of increasing the TMA concentration is the appearance of an extra system peak
that is very present in the entire chromatogram.
17
Effective
Gradient 2D
TMA in H2O/ACN
(95:5)(A)
0
50
180
100
0
50
100
150
200
250
Ammonium sulfate in
300 H2O/ACN (B)350
100
% Solvent
ACN/Buffer[A] (B)
H2O/ACN (A)
Effective Gradient 1D
80
60
40
20
0
160
140
120
Time (s)
100
80
60
40
20
0
% solvent
Figure 11: LC×LC-UV chromatogram of the separation of a mixture of 54 synthetic dye. Dual gradient analysis. First dimension: Strong Anion-Exchange,
Agilent PL-SAX 150x2.1nm, 8 µm, 0.01 mL/min. Second Dimension: Ion-Pair Chromatography, Agilent ZORBAX Eclipse Plus RRHT 50x4.6mm, 1.8 µm,
2.4 mL/min. Chromatogram recorded at 254 nm. Injection volume: 20.0 µL. Loopsize: 40 µL.
18
3.5 Hydrophilic Interaction Chromatography
HILIC was performed to see whether it would be a viable option for the 2-dimensional
chromatography. But the results showed that there was not enough interaction of the samples with
the HILIC column to establish enough retention for good enough separation. Thus HILIC was
discarded.
19
4. Conclusion and Perspectives
Anion-exchange, reversed-phase and HILIC columns were studied for the separation of old
synthetic dyes samples. None of these columns yielded full separation of all of the relevant synthetic
dyes. As a result, a study to the combination of two separation dimensions was initiated and a
comprehensive two-dimensional liquid separation system was successfully developed. The system
utilized an Agilent PL-SAX (150 mm x 2.1 mm, 8 µm) column for the first dimension and a Agilent
ZORBAX Eclipse Plus RRHT (50 mm x 4.6 mm, 1.8 µm) column for the second dimension. The
obtained peak capacity obtained by combination of these two columns was approximately 3000.
The LCxLC system was successfully used for the analysis and separation of 54 synthetic dyes
samples. By using this 2D-LC method the simultaneous separation of basic and acidic dyes was
established. It was furthermore concluded that using HILIC will not establish enough retention to be
a viable option for analysing these dyestuffs.
Now that a separation has been established the 2-D method is open to optimization. A possible
way to do this is by reducing the run-time even more, which is still over 5 hours with the current
method. This can be done by speeding up the gradients even more often, as there are still ‘empty
spaces’ left in the chromatogram. Other than this it can be relevant to investigate the ion-pair
effects more or even investigate alternative separation mechanisms, as RP and IEC are not the only
mechanisms applicable to these dyestuffs. Lastly, and most importantly, an important perspective of
this research, the original perspective in fact, is to do degradation studies. By manually degrading
the samples with UV-light for example, one can apply the developed 2-D method to analyse the
degradation.
20
5. References
[1]
Christie, R. (2014). Colour chemistry. Royal Society of Chemistry.
[2]
Erdmann, H. (1902). G. Schultz und P. Julius. Tabellarische Übersicht der künstlichen
organischen Farbstoffe. Vierte Auflage, herausgegeben von Dr. Gustav schultz. Berlin, R.
Gaertners Verlag (H. Heyfelder), 1902. Angewandte Chemie, 15(30), 767-767.
[3]
van Bommel, M. R., Berghe, I. V., Wallert, A. M., Boitelle, R., & Wouters, J. (2007). Highperformance liquid chromatography and non-destructive three-dimensional fluorescence
analysis of early synthetic dyes. Journal of Chromatography A, 1157(1), 260-272.
[4]
Dong, M. W. (2006). Modern HPLC for practicing scientists. John Wiley & Sons.
[5]
Snyder, L. R., Kirkland, J. J., & Dolan, J. W. (2010). Normal‐Phase Chromatography.
Introduction to Modern Liquid Chromatography, Third Edition, 361-402.
[6]
Barth, H. G., Jackson, C., & Boyes, B. E. (1994). Size exclusion chromatography. Analytical
chemistry, 66(12), 595R-620R.
[7]
Snyder, L. R., Dolan, J. W., & Gant, J. R. (1979). Gradient elution in high-performance liquid
chromatography: I. Theoretical basis for reversed-phase systems. Journal of
Chromatography A, 165(1), 3-30.
[8]
Cummins, P. M., Dowling, O., & O’Connor, B. F. (2011). Ion-exchange chromatography: basic
principles and application to the partial purification of soluble mammalian prolyl
oligopeptidase. In Protein Chromatography (pp. 215-228). Humana Press.
[9]
Jandere, P. (2007) Column Selection for Two-Dimensional LCxLC. LCGC Europe, 20(10) (pp.
510-525).
[10]
Cui, L., Wen, J., Zhou, T., Wang, S. and Fan, G. (2009), Optimization and validation of an ionpair RP-HPLC-UV method for the determination of total free iodine in rabbit plasma:
application to a pharmacokinetic study. Biomed. Chromatogr., 23: 1151–1159.
doi: 10.1002/bmc.1237
[a]
Dugo, P., Cacciola, F., Kumm, T., Dugo, G., & Mondello, L. (2008). Comprehensive
multidimensional liquid chromatography: theory and applications. Journal of
Chromatography A, 1184(1), 353-368.
[11]
Schoenmakers, P. J., Vivó-Truyols, G., & Decrop, W. M. (2006). A protocol for designing
comprehensive two-dimensional liquid chromatography separation systems. Journal of
Chromatography A, 1120(1), 282-290.
[12]
Stoll, D. R., Li, X., Wang, X., Carr, P. W., Porter, S. E., & Rutan, S. C. (2007). Fast,
comprehensive two-dimensional liquid chromatography. Journal of Chromatography A,
1168(1), 3-43.
[13]
Alpert, A. J. (1990). Hydrophilic-interaction chromatography for the separation of peptides,
nucleic acids and other polar compounds. Journal of chromatography A, 499, 177-196.
21
[14]
Society of Dyers and Colourists., & American Association of Textile Chemists and Colorists.
(1971). Colour index. Bradford.
[15]
Small, H. (2013). Ion chromatography. Springer Science & Business Media.
[16]
Hofenk de Graaff, J. H., Roelofs, W. G., & Bommel, M. R. V. (2004). The colourful past:
origins, chemistry and identification of natural dyestuffs. Archetype publications; AbeggStiftung.
[17]
Hemström, P., & Irgum, K. (2006). Hydrophilic interaction chromatography. Journal of
separation science, 29(12), 1784-1821.
22
Appendix 1: List of selection of synthetic dyes
Color Code
1735
2028
2030
3016
3034
3147
3203
3231
3289
3290
3408
3652
3712
3742
3824
3835
3855
4104
4328
4335
4412
4434
4518
4711
4958
4989
4995
5000
5027
5302
5305
5316
5347
5349
5365
5367
5528
5641
5706
6344
6531
6556
6709
Name
Indigo Carmine
Alizarine Red S
Indigotin
Orange II
Alizarine
Alizarin yellow
Diamond green B
Diamond green G
Chrysoidine
Azo Fuchsine 6B
Methyleen Blue
Auramine
Nigrosin
Crystal violet
Uranine A
Crystal ponceau 6R
Orange GG
Rhodamine B
Eosine A
Fuchsin
Chrysoin
Methyl Violet 2B
Water Blue IN
Rhodamine 6G
Croceine Orange G
Fast Red AV
Fast Red B
Ponceau RR
Azo Flavine 3 R
Patent Blue V
Erythrosine
Tartrazine
Metanil yellow
Amaranth
Flavazine L
Amido Naphtol Red G
Congo red
Yellowish light green SF
Brilliant Yellow
Ponceau 3RO
Quinoline Yellow
Cotton scarlet
Amido black 10B
C.I. Name
Acid Blue 74
Mordant red 3
vat blue 1
Acid orange 7
mordant red 11
Mordant yellow 1
basic green 4
basic green 1
basic orange 2
acid violet 7
Basic Blue 9
basic yellow 2
acid black 2
basic violet 3
acid yellow 73
acid red 44
acid orange 10
Basic Violet 10
Acid Red 87
basic violet 14
acid orange 6
Basic Violet 1
Acid Blue 93
Basic Red 1
Acid Orange 12
Acid Red 88
Acid Red 17
Acid Red 26
acid orange 1
Acid Blue 3
acid red 51
Acid Yellow 23
acid yellow 36
acid red 27
Acid Yellow 11
Acid Red 1
direct red 28
acid green 5
Direct Yellow 4
acid red 25
acid yellow 36
acid red 73
acid black 1
Mix
Charge proportion
24
11
0
4
11
0
4
11
1+
2
1+
4
1+
1
24
1+
1
0
1
?
4
1+
2
21
22
24
1-/1+
1
21
1+
4
11
1+
4
3-/1+
4
1+
1
11
12
24
24
14
?
2
21
34
14
31
14
24
24
3-/1+
4
24
22
0
4
24
24
23
6887
6923
6928
7088
7098
7177
7690
7759
7966
8511
8513
Orange IV
Naphthol yellow S
Orange I
Safranine T
Victoria Blue B
Fast acid magenta B
Wol red B
Martius yellow
Murexide
Vesuvine BA
Victoria Blue R
Acid Orange 5
acid yellow 1
Acid Orange 20
Basic Red 2
Basic Blue 26
acid red 33
acid red 115
acid yellow 24
Mordant Dye
Basic Brown 1
Basic Blue 11
1211+
1+
?
20
?
?
1+
2
4
1
1
2
4
4
1
4
4
2
24