optimization of a comprehensive two-dimensional …bachelor thesis chemistry optimization of a...

Bachelor Thesis Chemistry

Optimization of a comprehensive two-dimensional liquid

chromatography method for the separation of natural and

synthetic dyes

by

Lotje Bijkerk

27-06-2017

Studentnumber

10718826

Research institute

Van ’t Hoff Instituut of Molecular

Sciences

Research group

Analytical Chemistry

Supervisor

Dhr. prof. Dr. ir. P.J. (Peter)

Schoenmakers

Daily supervisor

Dhr. B.W.J. (Bob) Pirok MSc.

2 Bachelor Thesis Chemistry – Lotje Bijkerk – 27-06- 2017

Samenvatting

Het is plezierig om naar kunst te kijken, onder andere door de mooie kleuren die aanwezig kunnen

zijn in een kunstwerk. Al eeuwen lang maken mensen voorwerpen mooier door ze te kleuren. In de

loop der tijd is de manier van kleuren veranderd, evenals de kleurstoffen die gebruikt worden. Daar

waar er eerst alleen natuurlijke kleurstoffen werden gebruikt (die hun oorsprong vinden in plant- en

insectextracten), worden er sinds de toevallige ontdekking in 1865 van mauveïne, de eerste

synthetische kleurstof, voornamelijk synthetische kleurstoffen gebruikt. Synthetische kleurstoffen

worden door mensen gemaakt, en hun structuur wordt zodanig aangepast dat er een mooie, heldere

kleur ontstaat. Het maakproces van synthetische kleurstoffen is daarnaast minder arbeidsintensief

dan het extraheren van kleurstoffen uit natuurlijke bronnen. Maar kleuren vervagen in de loop der

tijd. De kleurstoffen worden blootgesteld aan bijvoorbeeld warmte en UV-licht (zonlicht). Deze

omstandigheden kunnen ervoor zorgen dat kleurstoffen degraderen, wat betekent dat de moleculen

van de kleurstoffen in kleinere stukjes breken en kapot gaan. Dit kan vervaging als gevolg hebben,

maar het is ook mogelijk dat de nieuwe kleinere moleculen een ander

soort kleur hebben. Dit roept de vraag op of de kleuren die wij in oude

schilderijen zien, wel de oorspronkelijke kleuren zijn. In Afbeelding 1 is

hier een voorbeeld van weergegeven. Aan de linkerkant van de afbeelding

is te zien wat de kleuren waren toen Van Gogh het schilderij

schilderde in 1888, en aan de rechterkant van de afbeelding zijn

de kleuren te zien die wij nu waarnemen wanneer we dit schilderij zien hangen in het museum. Dat

ziet er totaal anders uit!

Om deze reden is het belangrijk dat er onderzoek wordt gedaan naar kleurstoffen. Wanneer

je bijvoorbeeld een schilderij wil onderzoeken, wil je weten welke kleurstoffen er zijn gebruikt en of

deze kleurstoffen zijn gedegradeerd. Om dit te kunnen bepalen, moeten de moleculen van de

verschillende kleurstoffen gescheiden worden. Dit kan gedaan worden met vloeistofchromatografie.

Vloeistofchromatografie is een scheidingsmethode waarbij gebruik wordt gemaakt van een kolom

met een vaste fase waarmee verschillende moleculen verschillende interacties hebben. Zo kan

bijvoorbeeld gescheiden worden op lading (een molecuul van -1 zal langer blijven plakken aan de

vaste fase dan een molecuul van -2) of op hydrofobiciteit (een molecuul dat minder van water houdt

zal langer op de hydrofobe vaste fase blijven plakken dan een molecuul wat zich liever in de waterige

loopvloeistof bevindt). Omdat er eindeloos veel verschillende soorten kleurstoffen zijn, en er in een

schilderij ook een mix van natuurlijke en synthetische kleurstoffen kan voorkomen, is het gebruik

maken van één soort scheiding niet genoeg. Daarom wordt er in dit project gebruik gemaakt van een

scheiding in twee dimensies. Dit betekent dat er twee soorten scheidingen tegelijk (!) plaatsvinden.

Na elke kleine scheiding in de eerste dimensie wordt dit groepje gescheiden moleculen doorgegeven

naar de tweede dimensie waar deze moleculen dan op een zo anders mogelijke manier van elkaar

worden gescheiden. Dit zorgt ervoor dat er zoveel mogelijk verschillende soorten moleculen

gescheiden kunnen worden. Een twee-dimensionale methode opzetten is ingewikkeld. De methode

moet aan allerlei eisen voldoen, zoals dat er zoveel mogelijk scheiding moet zijn, het niet te lang mag

duren, maar nog belangrijker: dat de resultaten herhaalbaar zijn. In dit project is er daarom gefocust

op de optimalisering van een twee dimensionale methode om zowel natuurlijke en synthetische

kleurstoffen in één keer zo goed en zo herhaalbaar mogelijk te scheiden.

Afbeelding 1: 'De Slaapkamer' door Vincent van Gogh in 1888 (links) en 2015 (rechts).


Optimization of a comprehensive two-

dimensional liquid chromatography method

for the separation of natural and synthetic dyes

Abstract

The analysis of natural and synthetic dyes is increasingly a topic for studies because of the importance

of research on cultural heritage objects. Samples from cultural heritage objects (e.g. historic paintings

and textiles) can contain many different sorts of dyestuffs and their degradation products. When

using liquid chromatography as a separation method, the use of a two-dimensional setup can increase

the peak capacity and the number of sorts of dyestuffs that can be separated. In this project, a two-

dimensional method is optimized for the separation of a mix of 74 different natural and synthetic

dyestuffs. In the first dimension, strong anion exchange is used and in the second dimension reversed

phase is used, coupled together in a comprehensive 2D-LC method (LC x LC). In this project, it is kept

in mind while optimizing the LC x LC method that the method has to be compatible for a mass-

spectrometer. The mass-spectrometer can identify which dyestuff is present in which peak of the

chromatogram, when coupled to de LC x LC.


Table of contents 1. Introduction ............................................................................................................................................. 5

1.1. Dyestuffs ............................................................................................................................................ 5

1.2. Aim of project .................................................................................................................................... 6

2. Theory ....................................................................................................................................................... 7

2.1. Extraction and analysis .................................................................................................................... 7

2.2. Strong anion exchange ..................................................................................................................... 7

2.3. (Ion-pair) Reversed phase ............................................................................................................... 8

2.4. Comprehensive two-dimensional liquid chromatography............................................................ 9

2.5. Mass Spectrometry ......................................................................................................................... 11

3. Experimental .......................................................................................................................................... 12

3.1. Instrumental .................................................................................................................................... 12

3.2. Chemicals ......................................................................................................................................... 12

3.2. Analytical conditions ...................................................................................................................... 13

3.2.1. Sample preparation ................................................................................................................. 13

3.2.2. Methods .................................................................................................................................... 13

4. Results and discussion .......................................................................................................................... 16

4.1. Strong Anion Exchange .................................................................................................................. 16

4.2. Reversed Phase ............................................................................................................................... 20

4.3. LC x LC ............................................................................................................................................. 21

4.3.1. Long vs. short modulation time for the second dimension .................................................. 21

4.3.2. 50 vs. 75 mM ammonium sulphate in mobile phase B of the second dimension ............... 22

4.3.3. Step gradient in anion-exchange ............................................................................................ 23

4.3.4. Normal gradient in anion-exchange and 40% B in reversed phase .................................... 24

4.3.5. Step gradient in anion-exchange and 40% B in reversed phase .......................................... 25

4.3.6. Optimized LC x LC separation ................................................................................................. 26

4.4. Future perspectives ........................................................................................................................ 29

5. Conclusion .............................................................................................................................................. 30

6. Acknowledgments ................................................................................................................................. 32

7. References .............................................................................................................................................. 33

8. Appendix ................................................................................................................................................. 34


1. Introduction

1.1. Dyestuffs

Before the nineteenth century, objects were brightened up with

dyestuffs originating from natural sources (plants and insects).1

After the discovery of the first commercial synthetic dye

mauveine, in 1856 by William Perkin, the production of synthetic

dyes increased enormously.2 Mauveine itself had a short lasting

commercial lifetime, but its discovery let to the invention of a lot

of new and better dyestuffs.3 Nowadays there are over 400

synthetic dyes. The colouring matters are used in paintings,

textiles and food but also in quantitative and qualitative medical

analysis methods. The analysis of dyestuffs itself is gaining more

importance in the field of cultural heritage studies because it can

chart the originally used dyes and their degradation products.4

With a successful qualitative and quantitative method for the

analysis of the dyestuffs in cultural-heritage objects, it is possible

to gain knowledge about the original appearance and the creation-

process.5 A recent example in which dyestuff analysis can be of

great importance is the discovery of a 17th century dress in a

wreck in the Waddenzee (Figure 1). It is exceptional that such an

old piece of textile is found in a good state so it is the perfect

opportunity to gain more knowledge about the dyestuffs used in

that time. Besides the characterization of the used dyestuffs, the

degradation products can be studied to give an idea of what the

original state of the object looked like. In Figure 2 the painting ‘De

Slaapkamer’ by Vincent van Gogh is shown with its original

colours (left) and with the colours that are now visible (right). In

almost 130 years of existence, the used dyestuffs in the painting are degraded with visible different

colours as a result. In this case the analysis and the identification of the degradation products were

very useful to regain the original state of the painting.

Figure 1 : 17th century dress found in the Waddenzee.

Figure 2: 'De Slaapkamer' by Vincent van Gogh in 1888 (left) and 2015 (right).

Figure 3: influence of the use of different mordants to dyed textile.


As mentioned before, there are many different sorts of dyes. The dyestuffs differ in their

structure and can be acidic, basic, direct or mordant (reactive).5 The first three groups contain water-

soluble dyes, with the anionic and cationic dyes in the first two groups. Direct dyes can be used to

colour fibres without extra functional group in contrast to mordant dyes. A mordant is a metal that is

added to the dyestuff when the dyestuff itself has little or no affinity for the fibres.5 The metal forms

a coordination complex with the dyestuff and can be of influence for both the colour and the intensity

of the colour on a piece of textile. In Figure 3, the effect of mordants on different (natural) dyestuffs

on wool can be seen.

1.2. Aim of project

The analysis of natural and synthetic dyes is increasingly a topic for studies because of the importance

of research on cultural heritage objects. In most of these studies the dyestuffs are split up in sorts (e.g.

exclusively acidic or only sulfonated dyes) or only a few separate dyestuffs are studied.1,6,7,8,9 But as

said before, samples from cultural heritage objects can contain many different dyestuffs and possibly

also their degradation products. It is therefore necessary that an analysis method is found that is

usable for all dyestuffs and that needs as less sample as possible to keep the cultural heritage object

intact. Because of the fact that the samples can contain many different dyestuffs, there is chosen to

work with a two-dimensional setup. In this project both dimensions (strong anion exchange and ion-

pair reversed phase) were first optimized in a one-dimensional setup. Once both dimensions are

optimized apart from each other in separating megamix (a mix of 74 different natural and synthetic

dyestuffs), they were coupled together in a two-dimensional setup. From there on, the separation of

megamix is further optimized on the two-dimensional setup. The ultimate goal of this project is to set

up an optimized two-dimensional method which is compatible for the mass-spectrometer so that all

the small separated fractions can be transferred from the second dimension to the mass-spectrometer

to identify which dyestuff is present in which peak in the chromatogram.


2. Theory

2.1. Extraction and analysis

The analysis of dyes in historical objects is of great importance, but is also very difficult. First, the

number of possible dyes in an object is very high and together with a variety of degradation products

of all these dyes, many different products can be found in only one sample. Second, the dyes can be

impure (they were made for their bright colour instead of pure compounds) and chemically instable.5

Moreover, when researching a cultural heritage object, it is necessary to use as little as possible from

the object to prepare a sample so the integrity of the object is not disturbed.3 Therefore an effective

extraction method is needed to get the dyestuffs out of the object. For the extraction of dyestuffs from

wool there is already an effective method, in which only 1 mg wool is needed. The dyestuffs are

extracted with the method shown in Scheme 1 and dissolved in acetonitrile/DMSO (1:1).10 This

extraction method is set up to make analysis with high performance liquid chromatography and mass

spectrometry possible. These two analysis methods are an obvious choice because high performance

liquid chromatography combines reproducibility, speed and high resolution.1 Besides that, it can be

combined with analysis-systems for peak identifications such as mass spectrometry. In this project

both strong anion-exchange and ion-pair reversed-phase liquid chromatography are used. For

structure determination, it is possible to use mass spectrometry. In the next three sections, these

methods are further explained.

Scheme 1: extraction method for extraction of dyestuffs from wool.10

2.2. Strong anion exchange

In ion-exchange chromatography, molecules are separated on the basis of the affinity of the molecules

for the charged, stationary phase of the column.11 A separation is achieved between differently

charged molecules because of the different electrostatic interactions they have with the oppositely


charged stationary phase. Molecules containing more charged groups have more affinity to the

oppositely charged stationary phase and will thus have more retention. Ion-exchange

chromatography is a suitable method for the analysis of many synthetic dyes because the dyes vary

largely in charged states.5 In this study, anion exchange is used because most charged dyes contain a

negative group. The stationary phase for an anion-exchange column is positively charged, and so the

more negatively charged the analyte, the more retention. There are two different mechanisms for ion

exchange: weak and strong. In weak ion exchange the charge of the analytes, and with that their

affinity for the stationary phase, are pH dependent. The separation can only be performed in a narrow

pH range, while strong ion exchange can be performed within a wide pH range because the number

of charges on a strong ion exchanger is independent of the pH. With strong ion exchange, the affinity

of the analytes is determined by the concentration of salts from the mobile phase in the column. In

the gradients used while working with strong ion exchange, the concentration of salts is gradually

built up. This results in a competition mechanism in which the salt will replace the analytes in order

of the charge of the ions. Eventually all the analytes will lose their retention because an excess of salts

is being added. To give reproducible results, it is necessary to remove the (strong bonding) salts from

the stationary phase before analyzing a new sample. When injecting a new sample while the salts of

the mobile phase are still bonded to the stationary phase, the analytes will show less or no retention

because the stationary phase is still saturated with the salts of the mobile phase. Because of this

reason, a third mobile phase is added with an excess of weak interacting salts. While ‘flushing’ with

this mobile phase, the excess of salts from the third mobile phase will push the salts of the second

mobile phase away. Given the fact that it is a weak interacting salt, the column is easily re-equilibrated

with the first mobile phase (containing no salts). In section 3.2.2 and 4.1 the conditions needed for

good and reproducible anion-exchange results are further explained.

2.3. (Ion-pair) Reversed phase

Reversed phase HPLC is a form of high performance liquid chromatography in which molecules are

separated on the basis of hydrophobicity. A reversed-phase column is built up from a hydrophobic,

nonpolar (silica modified with octadecyl alkyl (C18) chains) stationary phase through which a polar

mobile phase flows. Hydrophobic molecules have higher affinity for the stationary phase than

hydrophilic molecules, resulting in a higher retention time.12 In this study, reversed phase is used

because dyestuffs contain conjugated ring structures, which are hydrophobic and thus give retention.

When focused on the degradation products of dyestuffs it is important to take in mind that the

degraded products will be smaller than the original dyestuffs and will thus be less hydrophobic. With


reversed-phase liquid chromatography, it is possible to demonstrate the degradation products

because of the difference in hydrophobicity. For these two reasons, reversed-phase liquid

chromatography is an obvious choice for the analysis of dyestuffs.

As mentioned before, dyestuffs can be separated by reversed phase, but because many

dyestuffs have a (negative) charge, it is important to use an ion-pair when practising reversed phase.

Because of the charge, the molecules can have hydrophilic interactions with the mobile phase, which

results in less retention with the stationary phase. When an ion-pair is used, the charged dyestuffs

are neutralized by a counter-ion (the ion-pair), which helps to create more retention. The fact that

besides reversed phase also ion exchange and (possibly) mass spectrometry are used as analysis

methods in the two-dimensional analysis, makes it more difficult to choose a suitable ion-pair. Firstly,

the ion-pair has to be low in UV absorption so it is not detected in the chromatogram.9 Secondly, the

ion-pair has to be volatile enough to be compatible with the mass spectrometer.9,13 The analysis of

aromatic sulfonated compounds (including azo-dyes) is widely studied.9,13,14 These studies show that

when using LC-MS methods, trialkylamines are possible ion-pairs. Both Fuh13 and Storm14 show that

triethylamine is a suitable ion-pair for the liquid chromatography analysis of highly sulfonated

compounds (azo dyes) and is volatile enough for the mass spectrometer when coupling the LC to the

MS.

2.4. Comprehensive two-dimensional liquid chromatography

As pointed out in the first section, the dyestuff samples are complex because one sample can contain

any number of different dyestuffs. Also, on the one hand, there is a lot of similarity between the

molecules within the groups of dyestuffs but on the other hand, there are great differences between

the different groups. This makes analyzing dyestuff samples difficult. Besides that, the concentration

of dyestuffs in the samples can be low because it is important to keep the objects of which the samples

are taken as intact as possible. These reasons indicate that analyzing dyestuff samples with a one-

dimensional method is not sufficient. Combining two liquid chromatography methods can increase

the possible number of analytes that are possible to separate in one time and can improve the peak

resolution and capacity.15 These advantages lead to the decision to work with a (comprehensive) two-

dimensional method when separating dyestuffs. In comprehensive liquid chromatography (LC x LC),

all effluent is transferred from the first-dimension column to the second-dimension column in small

volume fractions.15 After the analytes have passed through both columns, they reach the detector.

This results in a chromatogram in which series of individual chromatograms of the different fractions


that were passed on from the first dimension to the second dimension, are combined.15 The overall

chromatogram will thus span over the time of the first-dimension gradient. Because of this reason,

the first-dimension gradient is very slow, so that there is enough time for the peaks of the first

dimension to pass the effluent to the second dimension. This is done with the help of a switching

valve, built up from two loops: one that collects the effluent from the first dimension and one that

transfers its content to the second dimension. A schematic

overview of the switching valve is shown in Figure 4. The

valve switches in the modulation time: the time of the second-

dimension gradient. The time of the second-dimension

gradient is for this reason very short; to be sure all the small

fractions from the first dimension can be separated in the

second dimension.

When working with a two-dimensional setup, it is

important to keep the concept of orthogonality in mind.

Orthogonality is used, in separation science, as a measure of independency between separation

techniques.15 The peak capacity of a LC x LC chromatogram is higher when more orthogonal

mechanisms are used. In this project, a two-dimensional setup is made from strong anion exchange

(first dimension) and ion-pair reversed phase (second dimension). These separation mechanisms are

orthogonal to each other and are obvious choices to separate dyestuffs, as explained in the sections

above. This means that first the molecules will be separated on basis of their charge, from there the

differently charged groups will be further separated with ion-pair reversed phase. This will result in

a chromatogram in which hopefully all dyestuffs can be separated. As said in section 1.2, the ultimate

goal of this project is to set up an optimized two-dimensional separation method which is compatible

with the mass-spectrometer. When the molecules from all the different peaks from the second

dimension are send to the mass spectrometer, it is possible to identify the dyestuffs which are present

in the sample. Because of the fact that a two-dimensional setup gives better peak capacity and

therefore more separation is possible, mass spectrometry is easier to combine than when using a one-

dimensional setup. With a two-dimensional separation less dyestuffs will be present in one peak,

which results in a more pure fraction that will be passed on to the mass spectrometer. The mass-

spectrometer is then better capable to ionizing and detecting the molecules in the fraction. The

combination of a two-dimensional setup and mass-spectrometry will give a very strong indication of

the used dyestuffs in the samples.

Figure 4: Coupling of columns to the switching valve. (Source: Agilent OpenLAB CDS program).


2.5. Mass Spectrometry

Mass spectrometry is one of the most powerful analysis methods because it can give both quantitative

and qualitative information with high sensitivity.12 The mass spectrometer produces and weighs ions

from injected samples from which the molecular weight can be obtained.16 The molecules from the

sample are brought into the gas phase from where ions are produced. The (ion-)molecules are then

separated in their mass-to-charge ratio (m/z). The mass spectrometer contains an electrostatic field

which will accelerate the charged molecules. Thus the mass spectrometer is only capable of detecting

charged components.16 There are a few methods in which molecules are taken into the gas phase and

are ionized and there are also more possibilities in how the ions are then detected. The ions in this

study will be produced by electrospray ionization (ESI). With the ESI method, a small flow of liquid is

made into a fine mist, from which dissolved sample molecules are released from droplets by

evaporation of the solvent.16 As said in the last section, it is possible to couple the liquid

chromatographer to the mass spectrometer. In liquid chromatography, the sample is separated with

the help of a mobile phase, and the sample is therefore eluted in this mobile phase. Mass spectrometry

however, needs a sample as pure as possible. The solvent is evaporated, as indicated above, and it is

therefore necessary that the solvent (the mobile phase of liquid chromatography in this case) is

volatile enough for quick evaporation. It is also for this reason that the possible ion-pairs for the

reversed phase separation are limited. As said in the previous section, the ion-pair must be very

volatile when used in combination with mass spectrometry. After ionization, the molecules will be

detected with, in this study, a time-of-flight analyser. Ideally all ions have the same kinetic energy.

This will cause a separation between the mass of the ions because of the kinetic energy equation Ekin

= ½ mv2. This equation shows that lighter molecules will have a higher velocity, so they will reach the

detector earlier than the heavier molecules.12

Mass spectrometry is a perfect (extra) analysis method after using two-dimensional liquid

chromatography because it can give information about which molecules are present in which peaks

of the chromatograms. This information is very useful when identification of used dyes and their

degradation products is needed.


3. Experimental

3.1. Instrumental

For the optimization of the gradients for the first and the second dimension, a one-dimensional setup

was used on a Shimadzu HPLC (HPLC 3). The machine was built up from a liquid chromatograph (LC-

10AD), a communication bus module (CMB-20A), an UV-VIS detector (SPD-20A), a mixer (FCV-10AL)

and a degasser (DGU-20A5). The system was controlled by the Shimadzu Real Time Analysis program

(LCsolution Version 1.21 SP1). The 1D-LC data was processed with Microsoft Office Excel 2013. The

two-dimensional setup was done on an Agilent 1290 Infinity 2D-LC system (Agilent, Waldbronn,

Germany). The machine was built up from an Agilent 1100 series autosampler, two Infinity 1290

binary pumps (G4220A), two Infinity 1290 thermostated column compartments (G1316C) and an

Infinity 1290 diode-array detector (G4212A) operated by an Max-Light Cartridge Cell (G412-6008, V0

= 1.0 µL). To switch from the first to the second dimension, a 2-position 8-port valve (G4236A) with

two 60-µL loops was used. The two- dimensional setup was controlled by the Agilent OpenLAB CDS

Chemstation Edition (Rev. C.01.04[35]) software. The 2D-LC data was processed with Transform.

The used strong anion exchange column was an Agilent PL-SAX column (PL1951-3802, 150 x

2.1 mm i.d., 8-µm particles, 1000-Å pore size). The used reversed phase column was an Agilent

ZORBAX Eclipse Plus C18 Rapid Resolution HT (959941-902, 50 x 4.6 mm, 1.8 µm particles, 1000-Å

pore size). In the one-dimensional setup, there was coupled an in-line filter (Agilent 1290 in-line

filter) to both the anion exchange column and the reversed phase column to protect the columns from

insoluble dyes. In the two-dimensional setup, the in-line filter was coupled to the first-dimension

column, the strong anion-exchange column.

3.2. Chemicals

The 74 authentic dyestuffs (natural and synthetic from the period 1850 – 1920) were obtained from

the reference collection of the Cultural Heritage Agency of the Netherlands (RCE, Amsterdam, The

Netherlands). It is possible that the samples contain impurities and degradation products. The

dyestuffs were dissolved in Acetonitrile (ACN, LC-MS grade, obtained from Avantor Performance

Chemicals, Deventer, The Netherlands) and DMSO (obtained from SAFC supply Solutions, Steinheim,

Germany). The mobile phases were prepared with in house purified milli-Q water (Millipore Q-POD,

Millipak Express, 0.22 µm), Acetonitrile (ACN, AR grade, obtained from Avantor Performance

Chemicals, Deventer, The Netherlands). The salts and buffers used in the mobile phases for strong


anion exchange were ammonium sulphate (BioXtra ≥99.0%), sodium chloride (BioXtra ≥99.5%),

Trizma hydrochloride (BioPerformance Certified, cell culture tested, ≥99.0%) and Trizma Base

(Reagent grade; minimum ≥99.9%), all obtained from Sigma-Aldrich (Darmstadt, Germany). The

buffer and ion-pair for the mobile phases for reversed phase were prepared with formic acid (reagent

grade, ≥95%) and triethylamine (TEA, purity ≥99.5%), both obtained from Sigma-Aldrich (Darmstadt,

Germany).

3.2. Analytical conditions

3.2.1. Sample preparation

All optimization measurements are done with separation of megamix 60 ppm. This mix contains all

natural and synthetic dyestuffs from the period of (1850 – 1920), 74 dyestuffs in total, provided by

the Cultural Heritage Agency of the Netherlands. From each dyestuff a 5000-ppm solution of

ACN/DMSO 1:1 (v/v) was present, from which 60 ppm solution was made by putting together 100 µL

of each dyestuff with a supplement of ACN/DMSO 1:1 to 10 mL.

3.2.2. Methods

As written in section 2.1, the used column for anion-exchange liquid chromatography was an Agilent

PL-SAX. Two different gradient setups were used, both having the same mobile phases and ‘flush

gradient’ after the gradient for the peak area. For the one-dimensional setup, the flowrate was set to

1.0 mL/min. The injection volume was approximately 20 µL. The mobile phases consisted of (A) 60%

water, 40% ACN and 5 mM of Tris buffer (pH=7); (B) 60% water, 40% ACN, 5 mM of Tris buffer

(pH=7) and 50 mM ammonium sulphate and (C) 1M NaCl in water. The ‘flush gradient’ consisted of

15 column-volumes of C (5.25 min) followed up by 35 column-volumes of A (12.25 min). The first

gradient setup (for 1D measurements) had a time program of 31 min: 0 - 0.5 min isocratic at 100% A;

0.5 – 10.5 min a linear gradient to 100% B; maintained at 100% B for 2 min; 12.5 – 13.0 min a linear

gradient to 100% C; maintained at 100% B for 5.25 min; 18.25 – 18.75 min a linear gradient to 100%

A; maintained at 100% A for 12.25 min. The second gradient setup was a so-called step-gradient. The

gradient setup (for 1D measurements) had a time program of 22 min with a flow rate of 1.0 mL/min:

0 – 1.5 min gradually to 20% B; 1.5 – 2.2 min gradually from 20% to 60% B; 2.2 – 3.7 min gradually

from 60% to 100% B, maintained at 100% B for 0.1 min; 3.8 – 9.75 min isocratic at 100% C; 9.75 - 22

min isocratic at 100% A.


The used reversed-phase column was an Agilent ZORBAX Eclipse Plus C18 RRHT column

(section 2.1). For the one-dimensional setup, the flowrate was set to 1.0 mL/min. The injection volume

was approximately 20 µL. Two mobile phases were used: (A) 95% buffer, 5% ACN (v/v) and (B) 95%

ACN, 5% buffer. The buffer consisted of 5 mM triethylamine (TEA, ion-pair) in water, brought to pH=3

with formic acid. As with ion-exchange, two different gradient setups were used. The first gradient

setup (for 1D measurements) had a time program of 16 min: 0 – 0.5 min, isocratic at 100% A; 0.5 –

12.5 min a linear gradient to 100% B, maintained at 100% B for 0.5 min; 13.0 – 15.0 min a linear

gradient to 100% A, maintained at 100% A for 1 min. The second gradient setup (for 1D

measurements) also had a time program of 16 minutes, but started at 40% of mobile phase B: 0 – 0.5

min, 40% B; 0.5 – 12.5 min a linear gradient from 40% to 100% B, maintained at 100% B for 0.5 min;

13.0 – 15.0 min a linear gradient from 100% to 40% B, maintained at 40% B for 1 min.

For comprehensive two-dimensional liquid chromatography, the same anion-exchange and

reversed-phase columns were used with the same mobile phases. The injection volume was 10 µL.

The modulation time was 1.5 min. For the LC x LC measurements, the 1D gradients were modified

and translated to a 2D setup. Both the anion-exchange and the reversed phase gradients had to be

modified for two reasons. Translating the anion-exchange gradient from 1D to 2D (from a flowrate

from 1.0 mL/min to 0.02 mL/min) will result in an anion-exchange gradient that will take

approximately 11 hours, which is too long. The gradient is modified to a shorter one, which will take

approximately 3.5 hours. The reversed phase gradients had to be modified because of the modulation-

time in the 2D measurements. The modulation-time in the 2D setup is 1.5 min, a program time of 16

min is therefore not applicable. The reversed phase gradient is modified to a shorter one, so that it

fits in the modulation time of 1.5 min. The first dimension (anion-exchange) had a flowrate of 0.02

mL/min in the peak area and a flowrate of 1.0 mL/min in the flush gradient. The first anion exchange

(first dimension) gradient setup had a time program of 208 min: 0 – 5.0 min isocratic at 100% A (0.02

mL/min); 5.0 – 185 min gradually to 100% B (0.02 mL/min); 185 – 190 min gradually to 100% C

(0.02 mL/min); 190 – 195.75 min isocratic at 100% C (1.0 mL/min); 195.75 – 208 min isocratic at

100% A (1.0 mL/min). The step-gradient for anion exchange (first dimension) also had a time

program of 208 min: 0 – 5.0 min isocratic at 100% A (0.02 mL/min); 5.0 – 75.0 min gradually to 20%

B (0.02 mL/min); 75 – 110 min gradually to 60% B (0.02 mL/min); 110 – 185 min gradually to 100%

B (0.02 mL/min); 185 – 190 min gradually to 100% C (1.0 mL/min); 190 – 195.75 min isocratic at

100% C (1.0 mL/min); 195.75 – 208 min isocratic at 100% A (1.0 mL/min). The modulation time in

the 2D setup was 1.5 min, and the time program of the second dimension was therefore also 1.5 min.

The flow rate was set to 2.4 mL/min. One second dimension gradient started at 0% B: 0 – 1.20 min


gradually from 0% to 100% B; 1.20 – 1.30 to 100% A, maintained at 100% A till 1.50 min. The other

second dimension gradient started at 40% B: 0 – 1.20 min gradually from 40% to 100% B; 1.20 – 1.30

to 40% B, maintained at 40% B till 1.50 min.


4. Results and discussion

4.1. Strong Anion Exchange

The used gradient is built up from three different mobile phases as is explained in section 3.2.2. Mobile

phase C (1M NaCl in H2O) is used to remove the sulphate anions from mobile phase B (50 mM

(NH4)2SO4) from the positive stationary phase of the column. The NaCl is added in excess to be sure

that it is strong enough to remove all the sulphate anions even though the bond between the Cl anions

and the stationary phase of the column is not strong. To remove the excess of NaCl and to re-

equilibrate the column, mobile phase A (contains no salts) is used. To be sure the results are

reproducible, it is necessary to know how much column-volumes C and A are needed to bring back

the column to equilibrium. Firstly, the number of column-volumes C is optimized while the number

of column volumes A is held at a constant excess to be sure that only the influence of the quantity of

C is being shown. After the optimized number of column-volumes C, the number of column volumes

A is optimized while keeping the number of column-volumes of C constant at the optimized number.

This is schematically presented in Table 1- 3.

In Figure 5 the chromatograms of megamix with different gradients for the optimization of C

(Table 1 – 2) are shown. During the optimization of C, the number of column-volumes of A (for flushing

after C) is held constant at 40 column-volumes to be sure there is used enough of mobile phase A. The

measurements were performed in duplo to demonstrate if the gradients gave reproducible results.

The figure shows that gradient 5 had the best reproducible results. This shows that 15 column-

volumes of C works best for this column. During the optimization of the number of column-volumes

of A, therefore flushing with mobile phase C will be held constant at 15 column-volumes.

Table 1: strong anion exchange gradient setup with variables X, Y and Z (see Table 2 and Table 3).

Time A (%) B (%) C (%)

0 100 0 0

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12.5 0 100 0

13.0 0 0 100

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Table 2: schematic overview of optimizing number of column volumes of A with the variables X, Y and Z (Table 1).

Gradient Column-volumes of C X Y Z

1 35 25.25 25.75 39.75

2 30 23.50 24.00 38.00

3 25 21.75 22.25 36.25

4 20 20.00 20.50 34.50

5 15 18.25 18.75 32.75

Figure 5: Megamix on strong anion exchange with different gradients for the optimization of column-volumes of mobile phase C. Used column is Agilent B.041. Specifications of the used gradients are given in Table 1 - 2.


The gradients that were less reproducible (gradients 1 - 4) than gradient 5, show differences in peaks

in the area of the mono-anions. This can be explained by the fact that the salt used in mobile phase B

and C, ammonium sulphate and sodium chloride, have a -1 charge. The mono-anions of the sample

have to compete with these chloride anions directly from the moment of injecting when too much

column volumes of C is used. The mono-anions of the sample will have less retention because the

chloride anions are taking their place on the column. There is less difference in the peak area of the

di-anions of the sample because they will bind stronger to the stationary phase of the column than

the chloride anions. This results in less competition and therefore a smaller difference in the retention

time is seen when more or less sodium chloride is used.

In Figure 6 the chromatograms of megamix with different gradients for the optimization of A

(Table 1 and Table 3) are shown. Gradient 6 shows the best reproducibility and the best divided peaks.

In gradient 6 there is flushed with 15 column-volumes of C and 35 column-volumes of A (Table 3).

These numbers of column volumes will be used for flushing after each ion-exchange measurement to

be sure that the ion-exchange column is fully re-equilibrated and that the results are reproducible.

Table 3: schematic overview of optimizing number of column volumes of A with the variables X, Y and Z (Table 1).

Gradient Column-volumes of A X Y Z

6 35 18.25 18.75 31.00

7 30 18.25 18.75 29.25

8 25 18.25 18.75 27.50

Figure 6: Megamix on strong anion exchange with different gradients for the optimization of column-volumes of mobile phase A. Used column is Agilent B.041. Specifications of the used gradients are given in Table 1 and Table 3.


In the 2D setup, the flowrate of the first dimension (ion-exchange) is very slow (0.02 mL/min).

It therefore takes a long time to use the gradient of B which is used in the optimization gradients. The

time of a 2D setup would take approximately 11 hours when the optimized gradient is used. It is better

and more sustainable to have a shorter gradient. For this reason the very first 2D measurements were

done with the gradient for B which was used before in this research.5 Mobile phase B gradually goes

up to 100% in 180 minutes, which corresponds with 5.14 column-volumes. The results of this 2D

setup were not reproducible. When the used gradient was translated back to a 1D setup with a flow

of 1.0 mL/min, it showed that the analytes with a charge of -2 did not come off the column with this

little amount of column-volumes of B. This is shown in Figure 7 (left). The molecules with a di-anionic

charge have a retention time of approximately 6 minutes, while C is added after 4.5 minutes. This

explains why the results in the 2D setup were not reproducible. Because the time is wanted to be as

short as possible, another solution had to be found other than a longer gradient for B. For this reason,

it is tested if increasing the concentration of ammonium sulphate in mobile phase B helps to get all

the analytes off the column in a limited time. Three different concentrations were tested (50 mM, 75

mM and 100 mM) in the translated gradient from the 2D setup. In Figure 7 the results of 50 mM (left)

and 75 mM (right) ammonium sulphate are shown. With a concentration of 75 mM ammonium

sulphate, all the analytes come off the column before flushing with C. The 100 mM solution gives

better separated peaks (not shown here), but because of the danger of clogging and the possible use

of a mass spectrometer, there is chosen to work with 75 mM ammonium sulphate in mobile phase B.

When a new Agilent column (B.048) was used, the 50 mM ammonium sulphate in mobile phase B

gave better results. In Figure 8 the translated gradient on the new Agilent column is shown with 50

mM (left) and 75 mM (right) ammonium sulphate. There is thus worked with 50 mM mobile phase

because it gave better and more reproducible results (Figure 8) and because an as low as possible

concentration of ammonium sulphate is preferred to prevent clogging and to take into account the

possible use of a mass-spectrometer. From these results, it can be stated that a higher concentration

of ammonium sulphate in mobile phase B is needed when the column is older and in a less good state.

Figure 7: Megamix with translated 2D ion-exchange gradient with 50 mM (left) and 75 mM (right) ammonium sulphate in mobile phase B on Agilent Anion Exchange column B.041.


4.2. Reversed Phase

The used reversed phase gradient on 1D is based on the article of Pirok et al.5 The mobile phases used,

contained triethylamine (TEA) as an ion-pair instead of TMA or TBA because TEA is more compatible

with the mass-spectrometer.13,14 Two different gradients were tested for the reversed phase

separation of megamix. The first one tested, starts at 100% mobile phase A (95% buffer and 5% ACN)

and 0% of mobile phase B (95% ACN and 5% buffer).5 The second gradient tested starts at 40%

mobile phase B. It is chosen to test the second gradient for two reasons. The first reason is that the

first gradient shows no peaks before the gradient is at 40% B, so starting at 40% B will ensure that

the peaks will come earlier in the gradient. The second reason is that the ‘breakthrough effect’ will be

reduced in the 2D setup when the second dimension (reversed-phase) will start at 40% of mobile

phase B because the mobile phase used in the first dimension (anion-exchange) also consists of 40%

acetonitrile. The fractions of the first dimension are injected into the second dimension and the

concentration of ACN in the mobile phase used in the first dimension is much higher than in the

mobile phase of the second dimension. The analytes will than rather retain in the solution of the first

dimension than in that of the second dimension, which results in less retention in the second

dimension. This breakthrough effect is reduced when an injection solvent is used that is as weak an

eluent as possible.17 The use of a gradient that starts at 40% of mobile phase B in the second

dimension is therefore a logical choice. The chromatograms of both gradients in a one-dimensional

setup are shown in Figure 9 and Figure 10. The peaks in the gradient that starts at 40% B (Figure 10)

come earlier than the peaks in Figure 9. The reason for this effect is explained above. An important

thing to take into account when the peaks shift to a shorter retention time is that degraded dyestuffs

are less hydrophobic and will therefore have a shorter retention time. For this reason, the shift cannot

Figure 8: Megamix with translated 2D ion-exchange gradient with 50 mM (left) and 75 mM (right) ammonium sulphate in mobile phase B on Agilent Anion Exchange column B.048.


be too great because otherwise the degraded products will disappear in the t0 peak. The effect of the

(possibly) reduced breakthrough effect can only be seen on 2D measurements.

Figure 9: Megamix on reversed phase with the gradient starting at 0% of mobile phase B. (see section 3 for specifications of gradient and mobile phase).

4.3. LC x LC

The aim of this project is to optimize the 2D method used in the publication of Pirok et al.5 Besides

the optimization of the method, it is the aim to be able to separate more dyestuffs (74 instead of 54),

a mix of natural and synthetic dyestuffs instead of only synthetic dyes, and to make the method

reproducible. The 2D chromatogram of their research is shown later in this section. After the separate

optimization of the first and second dimension, different 2D setups were tested and they are

compared to see which gradient works best for the separation of megamix (74 different synthetic and

natural dyestuffs). In the first dimension (strong anion exchange) the re-equilibration of the column,

the mobile phases and the gradient were optimized in a one-dimensional setup. Furthermore, the

second-dimension gradients (ion-pair reversed phase) were tested on good separation and

reproducibility in a one-dimensional setup. With these optimized dimensions, different combinations

were tested in a two-dimensional setup. In the next sections, the chromatograms of the different

gradients are shown and compared to each other. Finally, the best tested gradient is compared to the

chromatogram in the article of Pirok et al. to show the improvements.5

4.3.1. Long vs. short modulation time for the second dimension

Firstly, it is tested if it is better to use a longer (3 minutes) or shorter (1.5 minutes) modulation time

for the second dimension. A longer modulation time could improve the separation in the second

dimension because the reversed phase gradient is more spread out. A disadvantage of a longer

modulation time is the fact that bigger fractions of the first dimension will be send to the second

dimension. This means that obtained separation in the first dimension will be partly lost when

Figure 10: Megamix on reversed phase with the gradient starting at 40% of mobile phase B. (see section 3 for specifications of gradient and mobile phase).


sending it to the second dimension. In Figure 11 the chromatogram of the separation of megamix is

shown with a modulation time of 3 minutes and in Figure 12 the chromatogram with a modulation

time of 1.5 minutes is shown. The modulation time of 1.5 minutes shows more separation between

the di- and tri- anionic molecules but the separation between the mono-anionic molecules is not very

different apart from the fact that all peaks in in the chromatogram in Figure 11 are more intense and

broader. This effect is seen in more chromatograms, but it is still unclear what the cause for this

problem is. Because there is more separation when a shorter modulation time (1.5 minutes) is used,

there is chosen to work with a modulation time of 1.5 minutes (90 seconds) in the following

chromatograms.

4.3.2. 50 vs. 75 mM ammonium sulphate in mobile phase B of the second dimension

As described in Section 3.1, the concentration of ammonium sulphate in mobile phase B of anion-

exchange (60% water, 40% ACN, 5 mM tris buffer pH=7 and a to be determined mM of ammonium

sulphate) has influence on the separation of natural and synthetic dyes. On the first used Agilent anion

exchange column (B.041), 50 mM of ammonium sulphate was not enough for all analytes to lose their

retention (Figure 7) in the relatively short time when the 2D gradient is translated to a 1D gradient.

When 75 mM ammonium sulphate was used, all the analytes lost their retention and the result was

reproducible (Figure 7). When switched to a 2D setup, a new Agilent anion-exchange column was

used (B.048) and therefore both mobile phases were tested again (Figure 8). In Figure 13 and Figure

14, the 2D results of these measurements are shown. When 50 mM ammonium sulphate is used, more

peaks were obtained. For this reason, and also because as less ammonium sulphate as possible is

Figure 11: UV chromatogram (254 nm) of LC x LC (1st dimension strong anion exchange and 2nd dimension reversed phase) separation of megamix 60 ppm with a modulation time of 3 minutes in the second dimension.

Figure 12: UV chromatogram (254 nm) of LC x LC (1st dimension strong anion exchange and 2nd dimension reversed phase) separation of megamix 60 ppm with a modulation time of 1.5 minutes in the second dimension.

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wanted when the LC x LC is coupled to the mass-spectrometer, there is chosen to work with 50 mM

ammonium sulphate in mobile phase B of anion exchange.

4.3.3. Step gradient in anion-exchange

As described in section 3.2.2 a step-gradient for the second dimension is tested. In this step- gradient,

the time to go from 0% to 20% of mobile phase B is spread out, the time from 20% to 60% is speeded

up and the time to go from 60% to 100% is again spread out. This setup is chosen because most

analytes lose retention between 0% and 20% and between 60% and 100% of mobile phase B. In the

time between 20% and 60% there are two long peaks, which could be shortened with a steeper

gradient. In Figure 15 the 2D result of a step gradient in the first dimension is shown. This

chromatogram indeed shows shorter peaks in the time between 20% and 60% of mobile phase B. The

peaks in the other areas are not better separated, but the first peak in the first dimension is shifted

from 25 to 30 minutes. This is a result of the less steep gradient between 0% and 20% of mobile phase

B. This shift is useful because it shows that it is possible to have more separation between the analytes

with a neutral or positive charge and (almost) no retention. In the next sections both the normal and

step gradient are therefore used to compare if a step gradient in combination with different reversed-

phase gradients works better than a normal gradient combined with the same tested reversed-phase

gradients.

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Figure 13: UV chromatogram (254 nm) of LC x LC (1st dimension strong anion exchange and 2nd dimension reversed phase) separation of megamix with mobile phase B in the 1st dimension containing 50 mM ammonium sulphate). See section 3.3.2 for specification of the gradient.

Figure 14: UV chromatogram (254 nm) of LC x LC (1st dimension strong anion exchange and 2nd dimension reversed phase) separation of megamix with mobile phase B in the 1st dimension containing 75 mM ammonium sulphate). See section 3.2.2 for specification of the gradient.

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4.3.4. Normal gradient in anion-exchange and 40% B in reversed phase

As described in section 3.2.2 and section 4.2 there are two different reversed phase gradients used. In

the first gradient, there is started with 0% of mobile phase B (95% ACN and 5% buffer) whether in

the second gradient there is started with 40% of mobile phase B. The 2D result of this reversed phase

gradient in combination with the ‘normal’ anion exchange gradient is shown in Figure 16. In this

chromatogram the reversed-phase peaks (second dimension) come earlier, which fill up the loose

space (0 – 40 seconds) which is present in the 2D chromatograms in which there is started with 0%

of mobile phase B (Figure 13 and Figure 14). In this chromatogram, all peaks are more intense and

broader for a still unknown reason. Due to this, it is not possible to accurately compare the degree of

breakthrough effect in the gradient which starts at 0% and the gradient which starts at 40%.

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Figure 15: UV chromatogram (254 nm) of LC x LC (1st dimension strong anion exchange and 2nd dimension reversed phase) separation of megamix with a step gradient in the 2nd dimension). See section 3.2.2 for specifications of the gradient.

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Figure 16: UV chromatogram (254 nm) of LC x LC (1st dimension strong anion exchange and 2nd dimension reversed phase) separation of megamix with the 2nd dimension gradient starting at 40% mobile phase B (95% ACN and 5% buffer)). For specifications of the gradient see section 3.2.2.


4.3.5. Step gradient in anion-exchange and 40% B in reversed phase

The last combination tested, is a combination of the step-gradient in the first dimension and starting

at 40% of mobile phase B in the second dimension. The result of this combination is shown in Figure

17. The first peak in the first dimension is again shifted to 30 minutes, and more separation between

the neutral and positive charged molecules (in t0) is obtained in reversed phase. The reversed phase

peaks are starting at 5 seconds instead of 40 seconds, so the separation in reversed phase is more

spread out. Also the peaks between 20% and 60% of mobile phase B in the first dimension are

shortened. This combination of gradients can be seen as an improvement in many prospects for the

above named reasons. But also in this chromatogram the peaks are more intense and broader for an

unknown reason, which results in the fact that this chromatogram is not good enough to accurately

compare to the chromatogram shown in Figure 12 (normal 1st dimension gradient and starting at 0%

B in the 2nd dimension). The peaks were too intense, which results in a ‘cut off’ of the peaks in

translating the signals into a 2D chromatogram. The peaks are therefore blunt instead of sharp, and

in the 2D chromatogram this results in thick, broad and intense signals. A few options that can cause

the bad condition of the peaks are excluded by testing in a one-dimensional setup. The reversed-phase

column (Agilent ZORBAX C-18) was still in good shape and gave the same separation of megamix as

earlier measurements. Moreover, the newly made megamix was tested and showed no differences

with the former megamix. There were made new mobile phases in cleaned bottles. None of these

variables had influence on the intensity and the broad of the peaks in the two dimensional

chromatograms. In future research, it could be interesting to test this combination of gradients again

when the shape of the peaks is recovered, because the combination looks promising.

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Figure 17: UV chromatogram (254 nm) of LC x LC (1st dimension strong anion exchange and 2nd dimension reversed phase) separation of megamix with a step gradient in the 1st dimension and starting at 40% mobile phase B in the 2nd dimension). For specifications of the gradients and mobile phases see section 3.2.2.


4.3.6. Optimized LC x LC separation

After testing different gradients in both the first and second dimension and making different

combinations between them, the obtained chromatograms can be compared to the chromatogram

published by Bob Pirok et al.5 This chromatogram is shown in Figure 18. In Figure 19 the

chromatogram with the newly optimized gradient is shown. In this setup, the first dimension (anion

exchange) is built up with the first, normal, gradient described in section 3.2.2. Mobile phase B of the

first dimension contains 50 mM ammonium sulphate and the flushing gradient for re-equilibration

is used (section 4.1). In the second dimension, the gradient starts with 0% of mobile phase B from the

second dimension. The results that show the gradient that starts at 40% of mobile phase B were not

trustworthy because of too intense and broad peaks, so there is chosen to show the results that have

the gradient starting at 0% mobile phase B. Two different 2D chromatograms are shown with

different matrices. The first chromatogram (Figure 19) shows the results with matrix 1. This figure

clearly shows that it is possible to separate both natural and synthetic dyes in one sample. The

chromatogram shows a good separation between neutral, mono-, di- and tri- negatively charged

molecules. Besides that, there is more separation in the second dimension, reversed phase, within

these differently charged groups of molecules. Because of an optimized flushing gradient, the column

is re-equilibrated after each measurement, which results in more reproducible measurements. The

reproducibility of the optimized gradient is shown in Figure 20. In this figure, two measurements

using the same gradient and same sample are overlaid. The yellow/red signals show the first

measurement and the blue surrounded signals show the second measurement. The signals of the

neutral and positively charged molecules and the mono- and di- anionic molecules are perfectly

reproducible. The signals of the tri-anionic molecules show small differences. The good

reproducibility of the 2D measurements is a big improvement and is due to the optimized flushing

gradient for the first dimension (strong anion exchange).

In Figure 19, the UV chromatogram of the LC x LC separation of megamix is shown with the

optimized gradient. In the chromatogram, there are vertical blue (means low intensity) stripes visible.

These stripes appear when the matrix is moderated in a way that as much peaks as possible are

visible. When looked at the direct output of 2D signals in the OpenLAB CDS program, no differences

are spotted at the time where the blue lines appear in the transformed chromatograms. In Figure 21,

the UV chromatogram of the LC x LC separation of megamix is shown unedited. In this figure, the

stripes are less present and when observed good, no peaks disappear, but only the intensity of the

peaks is lower. The origin of the stripes is still unknown. The first possibility is that a few dyestuffs

are present in the megamix with an excessively high concentration, but when a new megamix with


extra checked proportions was prepared, the stripes were still there. Another possibility is that the

used mobile phases are contaminated, but after making new, clean mobile phases, the stripes were

still there. The stripes are however no interruption for the separation and the peaks in the

chromatogram are still visible because they have a much higher intensity than the stripes.

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Figure 18: UV chromatogram (254 nm) of LC x LC (1st dimension anion-exchange without optimized flushing gradient and 2nd dimension reversed phase with TMA as ion-pair) of 54 synthetic dyes. Modulation time of 2 minutes and injection volume of 20 µL. (Source: Pirok , B.W.J.; Knip, J.; Bommel, van, M.R. & Schoenmakers, P.J. Characterization of synthetic dyes by comprehensive two-dimensional liquid chromatograpy combining ion-exchange chromatography and fast ion-pair reversed-phase chromatography. J. Chromatogr. A, 1436, 144 (2016))

Figure 19: UV chromatogram (254 nm) of LC x LC (1st dimension strong anion exchange with optimized flushing gradient and 2nd dimension reversed phase with TEA as ion-pair) separation of megamix (74 natural and synthetic dyestuffs). See section 3.2.2 for specification of the gradients. The 2D chromatogram is made with matrix 1.


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Figure 21: UV chromatogram (254 nm) of LC x LC (1st dimension strong anion exchange with optimized flushing gradient and 2nd dimension reversed phase with TEA as ion-pair) separation of megamix (74 natural and synthetic dyestuffs). See section 3.2.2 for specifications of the gradients. The chromatogram is unedited (raw data).

Figure 20: Overlay of two different LC x LC UV chromatograms (254 nm) of the separation of Megamix (1st dimension strong anion

exchange with optimized flushing gradient and 2nd dimension reversed phase with TEA as ion-pair).


4.4. Future perspectives

As in all research, there are still many improvements possible. At first, the origin of the too intense

and broad peaks has to be discovered. Secondly, the origin of the vertical stripes in the

chromatograms has to be unmasked. When these two points are improved, it would be interesting to

test the combination of the step-gradient in the first dimension and the second dimension starting at

40% mobile phase B again. When a chromatogram is obtained with less intense and less broad peaks,

the combination of these gradients can be accurately compared to the one shown in Figure 21, which

was the best combination tested in this project. The combination of a step gradient in the first-

dimension and the second dimension starting at 40% mobile phase B looks promising because of the

possibility of less breakthrough effect and less loose space in the second dimension. Furthermore, the

long peaks in the first dimension are shortened when this combination is used.

Another very important subject for further research is the coupling of the mass spectrometer

to the LC x LC. In this project, there was not enough time to cover this coupling. Nevertheless, it is

always remembered when modifying the 2D method that it had to be compatible with the mass

spectrometer one day. When the mass spectrometer is coupled to the LC x LC, all peaks could be

identified and this would be a big reinforcement of the obtained data.

A step further in the separation of samples that contain both natural and synthetic dyes is the

separation of degraded samples. In Figure 22 a degraded megamix (4 hour radiation from a 365 nm

UV lamp) is separated in the best tested LC x LC method. This chromatogram shows a lot of separated

peaks, but as in more chromatograms, these peaks are too broad and too intense. In further research

a more optimized LCxLC method could be developed in which a degraded megamix can be fully

separated.

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Figure 22: UV chromatogram (254 nm) of LC x LC (1st dimension strong anion exchange and 2nd dimension reversed phase) separation of degraded megamix. See section 3.2.2 for specifications of the gradients.

30 Bachelor Thesis Chemistry – Lotje Bijkerk – 27-06-2017

5. Conclusion During this project, a two dimensional LC x LC separation of a mix of 74 natural and synthetic dyes

(megamix) was optimized. Firstly, both dimensions were optimized on a one-dimensional setup.

For the first dimension, strong anion exchange, the concentration of ammonium sulphate in

mobile phase B was optimized at 50 mM for the used Agilent SAX column (B.048). Furthermore the

re-equilibration of the column was optimized by creating a flushing gradient which was built up from

15 column-volumes of mobile phase C (1M NaCl in water) and 35 column-volumes of mobile phase A

(60% water, 40% ACN and tris-buffer to pH=7). The flushing gradient was done after each

measurement to be sure that the column was again in a good state and that the results were

reproducible. Two different gradients were tested: a ‘normal’ gradually gradient and a step gradient.

Both gradients showed reproducibility and were tested as functional in a two-dimensional setup.

With a step-gradient the loose space and long peaks in the chromatogram were decreased. Because

of too broad and too intense peaks in the 2D chromatogram in which the step gradient was used, it

was difficult to accurately compare the normal and step gradient. But both gradients looked

promising.

For the second dimension (ion-pair reversed phase) as well, two gradients were tested. The

difference between the gradients was the starting percentage of mobile phase B (95% ACN and 5%

buffer). In the first gradient, it started at 0% mobile phase B, while in the second gradient it started

at 40% mobile phase B. Increasing the starting percentage of B resulted in the peaks coming earlier

in the second dimension (and thus the peaks were more spread out) and probably led to less

breakthrough effect. This cannot be said with complete certainty because of the too broad and too

intense peaks in the 2D chromatogram.

The optimized dimensions with their different gradients were tested in several combinations

in a two-dimensional setup. The 2D chromatograms showed that a step-gradient in the first

dimension (strong anion exchange) shortened the long peaks in the zone of 20% to 60% of mobile

phase B. Moreover, the first peak shifted from 25 min to 30 min which was favourable. For the second

dimension (ion-pair reversed phase) starting at 40% of mobile phase B seemed to be of good

influence on the chromatograms. The combination of the step-gradient in the first dimension and

starting at 40% of mobile phase B in the second dimension therefore seemed like a promising LC x LC

method. But in the end, the comparison between the optimized method and the method used by Pirok

et al. was made with the combination of a normal gradient (with flushing gradient) in the first

dimension and starting at 0% mobile phase B in the second dimension (for specifications see section


3.2.2).5 This was done because most other chromatograms contained too broad and too intense peaks

and also showed vertical stripes. The chromatogram that was compared showed normal peaks and

was therefore more suitable for comparison. When the chromatograms were compared, it could be

concluded that the results were reproducible, that there was more separation in the optimized

method and that it was possible to separate both natural and synthetic in one sample.

The ultimate goal in this research is to develop a reproducible LC x LC – MS method, which

can separate and identify the molecules in a (degraded) dyestuff mix sample at the same time. In this

project, the coupling between the LC x LC and the MS is not yet accomplished, but steps were made in

the direction to a trustworthy and reproducible method.


6. Acknowledgments First of all, I would like to thank my daily supervisor Bob Pirok and his master students Sanne Berbers

and Noor Abdulhussain. I learned a lot from all their insights and advises. Together they ensured that

I critically thought through every step I took in this project. Secondly, I would like to thank Peter

Schoenmakers for the chance to do this bachelor project in the Analytical Chemistry group at the

University of Amsterdam. And last but not least I would like to thank my lab partners Nienke Meekel,

Laura de Wal and Ole Hofman for useful brainstorm sessions and for making my time on the lab even

more fun.


7. References (1) Wouters, J. Stud. Conserv. 1985, 30 (3), 119–128.

(2) Lye, J. Colour chemistry; 2002; Vol. 27.

(3) Bommel, M. R. Van; Vanden, I.; Wallert, A. M.; Boitelle, R.; Wouters, J. 2007, pp 260–272.

(4) Confortin, D.; Neevel, H.; Brustolon, M.; Franco, L.; Kettelarij, A. J.; Williams, R. M.; van

Bommel, M. R. J. Phys. Conf. Ser. 2010, 231 (1), 12011.

(5) Pirok, B. W. J.; Knip, J.; van Bommel, M. R.; Schoenmakers, P. J. J. Chromatogr. A 2016, 1436,

141–146.

(6) Holčapek, M.; Jandera, P.; Zderadička, P. J. Chromatogr. A 2001, 926 (1), 175–186.

(7) Novotná, P.; Pacáková, V.; Bosáková, Z.; Štulík, K. J. Chromatogr. A 1999, 863 (2), 235–241.

(8) Serrano, A.; Van Bommel, M.; Hallett, J. J. Chromatogr. A 2013, 1318, 102–111.

(9) Socher, G.; Nussbaum, R.; Rissler, K.; Lankmayr, E. J. Chromatogr. A 2001, 912, 53–60.

(10) Bijkerk, L.; de Wal, L. Extractie en analyse van kleurstoffen, 2017.

(11) Fritz, J.; Gjerde, D. Instrumentation 2000, 59 (4).

(12) Harris, D. C.; Lucy, C. A. Quantitative Analytical Chemistry, 9th revised edition; W.H. Freeman

& Co Ltd, 2015.

(13) Fuh, M. R.; Chia, K. J. Talanta 2002, 56 (4), 663–671.

(14) Storm, T.; Reemtsma, T.; Jekel, M. J. Chromatogr. A 1999, 854 (1–2), 175–185.

(15) Jandera, P. Cent. Eur. J. Chem. 2012, 10 (3), 844–875.

(16) Williams, D.; Fleming, I. Spectroscopic Methods in Organic Chemistry, sixth.; McGraw-Hill

Education: Berkshire, 2008.

(17) Jiang, X.; Van Der Horst, A.; Schoenmakers, P. J. J. Chromatogr. A 2002, 982 (1), 55–68.


8. Appendix

List of dyestuffs in megamix Sample number Trivial name Natural dyestuffs

0228 Sandelwood

0894 Kaempferol

0895 Emodin

1153 Brasilin

1154 Maclurin

1156 Turmeric

1164 Berberine

1166 Fisetin

1167 Rhamnetin

1168 Carminic acid

2029 Lawson

2032 Morin

2035 Quercetin

2036 Rutin

2961 Orcein

2989 Isatin

3177 Haematein

4796 (+)epicatechin

4799 (+)catehinhydrate

7200 Campechewood

Synthetic dyestuffs

1482 Silk scarlet N

1735 Indigo carmine

1763 Picric acid

2028 Alizarine Red S

2031 Indigotin

3016 Orange II

3034 Alizarine

3147 Alizarin yellow

3203 Diamond green B

3231 Diamond green G

3289 Chrysoidine

3290 Azo Fuchsine 6B

3408 Methylene blue

3652 Auramine

3712 Nigrosin

3742 Crystal violet


3824 Uranine A

3835 Crystal ponceau 6R

3855 Orange GG

4104 Rhodamine B

4328 Eosine A

4335 Fuchsin

4412 Chrysoin

4434 Methyl violet

4518 Water blue IN

4711 Rhodamine 6G

4958 Croceine Orange G

4989 Fast red AV

4995 Fast Red B

5000 Ponceau RR

5027 Azo Flavine 3 R

5302 Patent blue V

5305 Erythrosine

5311 Cochineal red A

5316 Tartrazine

5349 Flavazine L

5365 Metanil yellow

5367 Amido Naphthol red G

5528 Congo red

5641 Yellowish light green SF

5706 Brilliant yellow

6344 Ponceau 3RO

6531 Quinoline yellow

6556 Cotton scarlet

6709 Amido black 10B

6887 Orange IV

6923 Naphthol yellow S

6928 Orange I

7088 Safranine T

7098 Victoria blue B

7177 Fast acid magenta B

7690 Wol red B

7759 Martius yellow

7966 Murexide

8511 Vesuvine BA

8512 Wool/cloth scarlet

8513 Victoria blue R


2D gradient setups

Gradient 1e dimensie 2e dimensie 09062017_LOTJE_1 Normal 0 % B 09062017_LOTJE_2 Normal Longer modulation time (3 min.) 09062017_LOTJE_3 Step gradient 0% B 09062017_LOTJE_4 Normal Starting at 40% B 09062017_LOTJE_5 Step gradient Starting at 40% B

09062017_LOTJE_1 1e dimensie: ion exchange

Time A B C Flow 0.01 100 0 0 0.02 5.00 100 0 0 0.02 185.00 0 100 0 0.02 190.00 0 0 100 1.00 195.25 0 0 100 1.00 195.75 100 0 0 1.00 208 100 0 0 1.00

2e dimensie: reversed phase Time B 0.00 0.00 1.20 100.00 1.30 0.00 1.50 0.00


Time A B C Flow 0.01 100 0 0 0.02 5.00 100 0 0 0.02 185.00 0 100 0 0.02 190.00 0 0 100 1.00 195.25 0 0 100 1.00 195.75 100 0 0 1.00 208 100 0 0 1.00

2e dimensie: reversed phase with longer modulation time Time B 0.00 0.00 2.40 100.00 2.60 0.00 3.00 0.00

09062017_LOTJE_3 1e dimensie: step gradient ion-exchange

Time A B C Flow 0.01 100 0 0 0.02 5.00 100 0 0 0.02 75.00 0 20 0 0.02


110.00 0 60 0 0.02 185.00 0 100 0 0.02 190.00 0 0 100 1.00 195.25 0 0 100 1.00 195.75 100 0 0 1.00 208.00 100 0 0 1.00

2e dimensie: reversed phase Time B 0.00 0.00 1.20 100.00 1.30 0.00 1.50 0.00


Time A B C Flow 0.01 100 0 0 0.02 5.00 100 0 0 0.02 185.00 0 100 0 0.02 190.00 0 0 100 1.00 195.25 0 0 100 1.00 195.75 100 0 0 1.00 208 100 0 0 1.00

2e dimensie: reversed phase starting at 40% B Time B 0.00 40.00 1.20 100.00 1.30 40.00 1.50 40.00

09062017_LOTJE_5 1e dimensie: stepgradient ion-exchange

Time A B C Flow 0.01 100 0 0 0.02 5.00 100 0 0 0.02 75.00 0 20 0 0.02 110.00 0 60 0 0.02 185.00 0 100 0 0.02 190.00 0 0 100 1.00 195.25 0 0 100 1.00 195.75 100 0 0 1.00 208.00 100 0 0 1.00

2e dimensie: reversed phase starting at 40% B Time B 0.00 40.00 1.20 100.00 1.30 40.00 1.50 40.00

optimization of a comprehensive two-dimensional …bachelor thesis chemistry optimization of a...

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