Two Dimensional Liquid Chromatography Principles and Practical Applications 1st Ed 9789811561894 9789811561900 - Compress

SPRINGER BRIEFS IN MOLECULAR SCIENCE
Oliver Jones
Two-Dimensional
Liquid
Chromatography
Principles
and Practical
Applications
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Oliver Jones
Two-Dimensional Liquid
Chromatography
Principles and Practical Applications
123
Oliver Jones
Australian Center for Research on
Separation Science, School of Science
RMIT University, Bundoora West Campus
Bundoora, VIC, Australia
ISSN 2191-5407 ISSN 2191-5415 (electronic)

SpringerBriefs in Molecular Science
ISBN 978-981-15-6189-4 ISBN 978-981-15-6190-0 (eBook)
https://doi.org/10.1007/978-981-15-6190-0
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2020
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Singapore
This book is affectionately dedicated to my
wife Michelle. Not only a skilful and
compassionate leader in her own field of
computational chemistry but a perfect
companion for our joint journey in science.
Our marriage is far more than the sum of its
individual dimensions thanks to her.
Preface
The primary objective of this book is to identify and discuss the basic aspects of
two-dimensional chromatography (2DLC) and make them available in a simple and
easy to understand manner. An effort has been made to present useful information
on key topics such as column selection, solvent considerations, method develop-
ment, and data analysis. I have not attempted to completely cover the subject but
rather to provide a basis for understanding key concepts, and provided references so
that a keen reader can further his or her study as needed. While some familiarity
with the terminology of chromatography and analytical chemistry is assumed in
places, I have tried to keep jargon and mathematical formulae to a minimum.
Should any readers want to brush up on HPLC theory, I highly recommend the
excellent CHROMacademy website (http://www.chromacademy.com/). It covers a
huge range of information on everything chromatography related. It includes some
great tutorials, application notes and study guides. You can get five years of free
access if you work or study at a university.
I feel that 2D is coming of age and is now far easier to use than many chemists
(and if you are reading this you are a chemist, at least in part) realise. I hope this
book will go some way towards confirming that feeling and encourage more sci-
entists to use the technique.
The book is a result of my teaching and postgraduate supervision in both gas and
liquid chromatography at RMIT University in Melbourne over the last eight years.
I thank all the students I have had the pleasure of working with during this time; this
work would not have been possible without them. I’d also like to acknowledge all
the help and support I have received from industry partners at Agilent, Shimadzu,
and LECO who have all provided, and continue to provide, superb technical help
and thoughtful conversations.
Melbourne, Australia Oliver Jones
vii
Acknowledgements
This book comes with thanks to three groups of peoples. Firstly, my family, friends,
and academic colleagues around the world who always support my endeavours.
Secondly, the citizens of Australia, particularly the state of Victoria, who employ
me to teach Chemistry to their sons and daughters. Finally, my fellow Australians
who support the Australian research enterprise with their taxes through agencies
such as the Australian Research Council. I am sure most of them don’t know much
about separation science, or have even heard of it, but they helped this book come
to fruition just the same.
ix
Contents
1 Introduction to 2DLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2 Basic Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.2 Column Orthogonality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3 Peak Capacity and Undersampling . . . . . . . . . . . . . . . . . . . . . . . 16
2.4 Surface Coverage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.5 Gradient Elution Chromatography . . . . . . . . . . . . . . . . . . . . . . . 19
2.6 Solvent Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.7 Column Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.8 How to Use 2DLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3 Method Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2 What Factors Should I Consider? . . . . . . . . . . . . . . . . . . . . . . . 26
3.3 Offline Method Development . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.4 Online Method Development . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.5 Column Selection (Orthogonality) . . . . . . . . . . . . . . . . . . . . . . . 30
3.6 Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.7 Modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4 Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
4.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
4.2 Displaying the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.3 Algorithms and Processing Methods for 2DLC Data . . . . . . . . . . 43
4.4 In Silico Method Optimisation . . . . . . . . . . . . . . . . . . . . . . . . . . 45
xi
xii Contents
4.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
5 Hyphenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
5.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
5.2 Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
5.3 Multiple Chromatographic Methods . . . . . . . . . . . . . . . . . . . . . . 53
5.4 Combining Chromatography and Electrophoresis . . . . . . . . . . . . 54
5.5 LC GC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
5.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
6 Applications of 2DLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
6.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
6.2 Pharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
6.3 Natural Product Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
6.4 Metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
6.5 Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
6.6 Lipidomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
6.7 Environmental Science . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
6.8 Forensic Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
6.9 Polymer Science . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
6.10 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
7 Conclusions and Future Developments . . . . . . . . . . . . . . . . . . . . . . . 71
7.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
7.2 3DLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
7.3 Column Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
7.4 Miniaturisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
About the Author
Oliver Jones is an analytical chemist based at RMIT University in Melbourne,

where he holds the rank of professor and serves as the Associate Dean of
Biosciences and Food Technology. Originally from Manchester in the UK, Oliver
was awarded a B.Sc. (Honours) from Queen Mary University of London and
obtained his M.Sc. and Ph.D. from Imperial College London. He then held a
Postdoctoral Fellowship at the University of Cambridge. Oliver left Cambridge to
take up a lectureship at the University of Durham before moving to RMIT in 2012.
Oliver’s group conducts research in analytical methods and technologies, pre-
dominantly in multidimensional chromatography and NMR, for a range of appli-
cations, particularly metabolomics and the trace analysis of environmental
pollutants. Oliver also has a passion for teaching and recently helped develop a
mobile game app called “Chirality-2” to help teach organic chemistry. He is cur-
rently a member of the Australian Academy of Science National Committee for
Chemistry.
xiii
Abbreviations
1D One dimension(al)
1DLC One-dimensional high-performance liquid chromatography
2D Two dimension(al)
2DLC Two-dimensional high-performance liquid chromatography
ACN Acetonitrile
AEX Anion exchange chromatography
AMDE Absorption distribution, metabolism and excretion
ANP Aqueous normal phase
API Active pharmaceutical ingredients
ASM Active solvent modulation
C18 Octadecylsilane
CCS Collision cross sections
CE Capillary electrophoresis
CN Cyanopropyldimethylsilane
DAD Diode array detector
DCM Dichloromethane
FID Flame ionisation detector
FT-IR Fourier transform infrared
GC Gas chromatography
GC GC Comprehensive two‐dimensional gas chromatography
HILIC Hydrophilic interaction chromatography
HPLC High-performance liquid chromatography
HR High resolution
IC Ion chromatography
ID Internal diameter
IEX Ion exchange chromatography
IMS Ion mobility spectrometry
KOH Potassium hydroxide
LC Liquid chromatography
LC LC Comprehensive two‐dimensional liquid chromatography
xv
xvi Abbreviations
Mb Megabyte
MeOH Methanol
MPLC Medium pressure liquid chromatography
MS Mass spectrometry
NARP Non-aqueous reversed-phase chromatography
NH2 Amino
NMR Nuclear magnetic resonance
NP Normal phase
NPS Novel psychoactive substances
PARAFAC Parallel factor analysis
PCA Principal component analysis
PFP Pentafluorophenyl
PHPLC Preparative liquid chromatography
PLS Partial least-square regression analysis
QQQ Triple quadrupole
QToF Quadrupole time of flight
RP Reversed phase
SAX Strong anion exchange
SCX Strong cation exchange
SEC Size exclusion chromatography
SFC Super critical fluid chromatography
SPAM Stationary phase-assisted modulation
SPE Solid phase extraction
STAMP Separation technology for a million peaks
UV Ultraviolet
VEW Vacuum evaporation modulation
WAX Weak anion exchange chromatography
WCX Weak cation exchange chromatography
Chapter 1
Introduction to 2DLC
Abstract Today’s analytical chemists face increasing demands to maximize the

number of compounds that can be separated and identified in a single run but peak
overlap continues to be a problem in many chromatographic methods. One method
that might help to overcome these issues is multidimensional liquid chromatography,
which uses two columns of different phases. A sequential collection of aliquots is
made from the first column and reinjected onto a second; the resulting data are
then plotted in 2D or 3D space. The total peak capacity of such a system is the
combined peak capacities of each column. The ‘offline’ version of this technique,
using a fraction collector, was introduced over 40 years ago but with recent advances
in instrumentation and software, particularly the ‘online’ approach, using automated
switching valves, has led to increasing interest in the technique. Both offline and
online methods can be carried out as a comprehensive procedure, or via ‘heart-
cutting’, in which only specific peaks are analysed in the second dimension. Appli-
cations include proteomics, natural product chemistry, forensic science, and phar-
maceutical analysis. These successes are likely to be built on in the future as new
column chemistries and bio-informatic approaches are developed. In this chapter,
an overview of the two-dimensional liquid chromatography is presented to give the
reader a basic understanding of this emerging technology and its potential future
uses.
1.1 Background
Modern analytical chemistry is heavily reliant on both increasingly advanced analyt-

ical instrumentation and the equally increasingly advanced software needed to control
the instruments and process the resulting data (also increasingly complex). Never-
theless, due to the sheer diversity of compounds present in most modern samples,
and their correspondingly large range of physicochemical properties, it is difficult to
envisage a single analytical technique capable of analysing, let alone quantifying all
the possible chemical substances that might be present in a sample.
For these reasons, many studies make use of multiple analytical techniques, each
of which has advantages and disadvantages. Nuclear magnetic resonance (NMR)
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2020 1
O. Jones, Two-Dimensional Liquid Chromatography,
SpringerBriefs in Molecular Science,
https://doi.org/10.1007/978-981-15-6190-0_1
2 1 Introduction to 2DLC
spectroscopy is, for example, fast and reliable but lacks sensitivity. Gas chromatog-
raphy is sensitive and reproducible but is limited to compounds that are (or can
be made) volatile. Liquid Chromatography has good range, good sensitivity and can
potentially analyse almost any compound, but it is often hampered by peak resolution
issues. If one could increase the separation power of liquid chromatography while
retaining reproducibility and sensitivity one would have a very powerful analytical
tool. This book is intended as a primer on one such method—Two-dimensional liquid
chromatography (2DLC). Some familiarity with the instrumentation and techniques
of standard HPLC is assumed of the reader but jargon, mathematical equations, and
complex data analysis have been kept to a minimum.
Two-dimensional liquid chromatography is an interesting technique in that it is
both an old, and a young method. The basic principles have been around for over
40 years with the essential concepts being introduced in the late seventies (Erni and
Frei 1978). Comprehensive 2D-LC (LC × LC) as it is mainly thought of today (i.e.
where all of the effluent from the first column is passed to the second) was first
described in 1990 (Bushey and Jorgenson 1990). At this stage, however, the method
was restricted to specialists. Similar to the development of mass spectrometry (MS)
it wasn’t until advances in technology (primarily column and instrument hardware)
and software made it possible for a wide range of users to perform the very fast,
high-efficiency separations demanded by modern analysts that development really
started to pick up. In the early 21st century development had been pushed along to
the stage where LC × LC separations yielding peak capacities of up to 2,100 in
60 min were reported (Stoll et al. 2006). This has led to a large growth in the number
of papers in 2DLC over the last 20 years as shown in Fig. 1.1.
Fig. 1.1 Bar chart showing number of papers returned by year with the search term “two
dimensional AND liquid chromatography” at http://www.sciencedirect.com/ in March 2020
1.1 Background 3
Fig. 1.2 Schematic diagram of a 2DLC system
But let us take a step back and examine the basic principles of 2DLC. In principle,
it is not too different to standard, one dimensional HPLC. The extra hardware required
comes down to a second (binary) pump, (a third pump may be used as we shall see
in Chap. 3), some form of modulation device (e.g. a valve) to move the effluent
from column one onto column 2-either entirely (comprehensive) or in part (heart
cutting), and a second detector. Software to run all these systems smoothly and
efficiently as well as process and display the resulting data is also required. This may
be commercial software or custom-written code which is often written in MATLAB,
Mathematica or R.
A typical 2DLC set up is shown in Fig. 1.2. Here we can see that essentially a 1D
HPLC system plus another 1D HPLC system equals a 2D LC system.
Chemists and biochemists have actually been separating things in more than one
dimension for some time via two-dimensional electrophoresis or two-dimensional
thin layer chromatography for example. Even in modern HPLC there is more separa-
tion that just the chemical interactions in the column. A Diode array detector (DAD)
separates via wavelength for example and a mass spectrometer separates via mass
to charge ratio. The second dimension in 2DLC can be thought of as being like just
like 1DLC with an extra chemically selective detector. This can of course then be
extended with a further form of separation such as mass or wavelength.
The major advantage of 2DLC is the increase in separation space and resolving
power it gives to the user—and resolving power (in a reasonable time) is what it is
all about in analytical separation science (Poppe 1997). The total peak capacity of a
2DLC system is theoretically, but realistically not quite (as we will explore further in
Chap. 2), the product of peak capacities of the individual dimensions. The resulting
separation space far exceeds that of standard 1DLC systems, an illustration of this is
shown in Fig. 1.3. Additionally, when the column effluent from the first dimension
is focussed and re-injected onto the second for rapid analysis, the effects of peak
broadening can be greatly minimised.
2DLC comes in two flavours, offline and online; each brings slightly different
benefits and challenges. Online (automated) multidimensional chromatography
(either gas or liquid) involves coupling two columns, with uncorrelated retention
Fig. 1.3 Comparison of standard and comprehensive offline two-dimensional LC separation of

mushroom metabolites. Data are shown as both a contour plot (left) and a three-dimensional chro-
matogram (right). The white dots in the left panel represent detected peaks Based on data from
Pandohee et al. (2015)
mechanisms (e.g. polar and non-polar, or normal phase and reverse phase columns)
in series. During the analysis, a sequential collection of aliquots is made from the
first column (the first dimension) and reinjected onto a second column (the second
dimension) in multiple repeated alternating cycles via an automated switching valve.
Typically, an 8 or 10 port, two-position valve with two sampling loops is used. One
loop is connected to, and filled by, the first dimension effluent, while the contents
of the other loop are injected onto the second dimension column. The time between
each valve switch is referred to as the modulation time and this equals the analysis
time of the second dimension. The resulting data are then plotted in either 2D or
3D space. As we will see later, active modulation methods are an important part of
method sensitivity.
Offline 2DLC has been around for almost 40 years. It involves the use of a fraction
collector at the end of the first column to collect fixed aliquots of the eluent into
chromatography vials. The column of the unit can then be changed and each of the
previously collected fractions is then run as a ‘new’ sample. This method is relatively
simple and cheap to set up since all that is required is a standard one-dimensional
LC system, a fraction collector and appropriate data processing software. However,
since running multiple aliquots of multiple samples can add up to hundreds, if not
thousands of individual runs, the total analysis time for the offline technique is often
multiple days or weeks and is thus time and operator intensive.
1.1 Background 5
The use of longer columns in the second dimension can also cause samples to
become highly diluted so overloading of the column is sometimes necessary to ensure
a large enough signal is received by the detector.
Integrating the two resulting datasets from offline 2DLC can also be challenging
and require specialist knowledge as we shall see in Chap. 4. The left-hand image in
Fig. 1.3 for example, was generated using custom written code in Mathematica by Dr
Paul Stevenson (Stevenson and Guiochon 2013). It is also worth mentioning at this
point that LC × LC chromatograms look quite different to conventional LC profiles.
Instead of a series of peaks the data is plotted in a bidimensional plane where analyte
“spots” are scattered about, as can be seen in the left hand plot of Fig. 1.3—although
the data can also be plotted in a 3D format (right hand panel of Fig. 1.3).
Online 2DLC is easier to run and the high-pressure capability of modern columns
and instrumentation allows high flow rates and correspondingly fast analysis. Disad-
vantages included increased price and the fact that short second dimension columns
are required in automated 2DLC since the separation of a sub-sample in the second
dimension must be completed before the subsequent sub-sample from the first
dimension is injected.
The 2DLC approach can be divided further into two forms; comprehensive (LC
× LC) and heart cutting (LC-LC). In comprehensive LC × LC all of the effluent
from the first column is subjected to a second separation step. The second column
has a limited analysis time as each fraction has to be off the column before the next
fraction is injected to avoid mixing. This means the second column is usually run
under ultra-high pressure (UHPLC) conditions and this may complicate subsequent
MS hyphenation. It does however allow full characterisation of complex samples
and it is quite popular for such applications.
In heart-cutting 2DLC we only inject specific fractions of interest onto the second
column (this could be one or multiple fractions). Contemporary software packages
allow the user to select peaks of interest and automatic peak selection is also possible
using user-defined time windows. This is illustrated in Fig. 1.4.
The heart-cutting technique is often written as LC-LC to distinguish it from
comprehensive 2DLC (which is written as LC × LC). Similar terminology is also
employed in multidimensional gas chromatography.
The advantage with heartcutting is that a fraction(s) or peak(s) cut out from the
main sample can then be analysed with higher separation efficiency on the second
column as there are fewer competing peaks to worry about. If multiple cuts are made
the term multiple heart-cutting (mLC-LC) is used. This is a flexible and intuitive
method relative to comprehensive LC × LC and can be readily combined with Mass
Spectrometry (MS). It is popular for moderately complex samples and peak purity
assessments. Operated in a heart-cutting mode, 2D-LC can also be used a prepara-
tive technique (sometimes termed 2D-PHPLC). Here the first dimension essentially
serves as an extraction step, simplifying the sample matrix before separation of target
analyte(s) in the second dimension.
Fig. 1.4 Illustration of the principle of (multiple) heartcutting or (m)LC-LC, subjecting one or
more fractions of a chromatogram to a second separation mechanism
Fully automated 2DLC is a relatively new technique currently offered by systems

such as the Agilent 1290 Infinity (Fig. 1.5), Shimadzu Nexera-e and Waters
ACQUITY instruments. These instruments make use of multiple HPLC columns and
switching valves in conjunction with advanced software (to control both the valve
and the solvent pumps) to move eluent from the first column to the second automat-
ically. Such systems have only been around for the last five years or so but having
such off the shelf options has helped paved the way for the full exploration of 2D ×
LC, as has a coherent theoretical framework(s) to explain the reasons and principles
behind multidimensional LC separations (Giddings 1995; Pirok et al. 2019).
At this stage the reader may ask why we should care about increasing separations?
To answer this question, we only have to step into any modern laboratory and look at
the type of samples currently of interest to the modern-day chemist. Such samples are
often amazingly complex be they protein extractions, metabolites, water/soil samples
or food products. There is also a need to be able to measure more compounds, even
in targeted analysis. For example, not long ago an analyst looking as pesticides may
only need to test for 20–30 compounds, now they may need to look for 6000 or more.
There is therefore an increasing desire to expand the number of compounds that can be
separated in a single run. Although not yet widespread the increased separation power
of two-dimensional chromatography has great potential to meet this need in a large
variety of areas, particularly for the analysis of compounds that are too sensitive for
mass spectrometry but contained in matrices too complex for standard LC analysis.
1.1 Background 7
Fig. 1.5 Agilent 1290 2DLC system in the author’s laboratory at RMIT University in Melbourne
Australia
Such areas include, but not limited to, animal health, biomedicine, dairy science,
environmental toxicology, food science, functional genomics, pharmacology, plant
biology toxicology and the omic sciences.
The latest version of the Human Metabolome Database (version 4.0) for example
lists 114,100 individual entries (Wishart et al. 2018). This is, nearly a threefold
increase from version 3.0. This includes large quantities of predicted MS/MS and GC-
MS reference spectral data as well as predicted (physiologically feasible) metabolite
structures. Even this large database does not include all possible compounds/isomers.
This means that the actual number of human metabolites (both endogenous and
exogenous) could potentially be far higher. Typically, however, metabolomics studies
using standard commercial databases such as NIST and METLIN ‘identify’ only
around 100 metabolites.
In recent years, commercial libraries have been complemented by extensive
open-access databases, such as mzCloud and the Human Metabolome Database,
containing hundreds of thousands of spectra. The creation of in-house libraries of
pure compounds may be of help here but, of course, there can be no in-house libraries
made of, as yet, unknown metabolites. A range of in silico predictive tools have
emerged to assist with the interpretation of high-resolution MS data in this regard.
As yet none of these developments have led to thousands of metabolites per study
being identified routinely. Indeed, da Silva et al. (2015) estimated that in some cases
as few as 1.8% of the mass spectra obtained in metabolomics studies can be fully
identified and many features identified in mass spectra are listed only as ‘unknown
compounds’. Of course, one has to separate all of these compounds from each other
before one can identify them.
The growth and success of proteomics (the analysis of the entire protein comple-
ment of a cell, tissue, or organism under a specific, defined set of conditions) has
introduced a similar problem in that field. Current proteomic tools allow large-scale,
high-throughput analyses for the detection, identification, and functional investi-
gation of the least abundant as well as the most abundant proteins, as well as the
analysis of post-translational modifications such as glycosylation and phosphoryla-
tion. This vastly increases the number of sample components. In short people want
more samples analysis, in greater depth, faster.
The bottleneck in compound separation and identification is an issue that will
take the use of new research and analytical methods to solve. Recent developments,
such as HILIC (hydrophilic interaction liquid chromatography) (Creek et al. 2011)
and ANP (aqueous normal phase chromatography) (Pesek and Matyska 2012), have
greatly increased compound coverage in biological samples via liquid chromatog-
raphy, with the detection of over 1000 compounds being reported in certain sample
types (Callahan et al. 2009). This level of analysis is not present in most papers and,
since the theoretical maximum peak capacity for conventional liquid chromatography
is ~1500 (Guiochon 2006) even this method will soon reach its limit. The use of very
long columns is also required for such detailed analysis, meaning run times of hours
or even days are often required to attain such high values. Liquid chromatography
as a method can also still be hampered by lack of resolution. Some metabolites,
especially those present in low concentrations, may simply be crowded out and go
unseen. Many polar metabolites, such as sugars, and many amino acids are often
not retained by conventional reverse phase (RP) LC columns for example (Callahan
et al. 2009). In addition, the increased number of peaks results in correspondingly
crowded chromatograms, likely obscuring important (bio)chemical details.
The lack of ability to detect all metabolites present, and fully identify all metabo-
lites detected, termed the dark metabolome by Jones (2018), means that, despite
the great contribution of metabolomics to a range of areas in the last decade, a
significant amount of useful information from publicly funded studies is being lost
or unused each year. Similar problems exist for related areas such as proteomics
and pharmaceutical analysis as well as unrelated but equally important ones such
as polymer science. This loss of data limits our potential gain in knowledge and
understanding of important research areas such as cell biology, environmental pollu-
tion, plant science, food chemistry and health and biomedical research. Separation
science needs to develop new tools and methods for compound identification since
the separation of complex mixtures is of vital importance to modern chemistry.
With the above in mind, it can hopefully now be seen why 2DLC has such potential.
Indeed, new demonstrations of its capabilities appear almost weekly. That said, 2DLC
is not without its problems. Despite all its positive traits and increasing number of
applications and publications 2DLC has struggled to break into the academic or
industrial laboratory as a routine analytical technique. Issues such as mobile phase
compatibility issues, column orthogonality, band broadening and data analysis all
need to be dealt with before this can happen. These issues are discussed in subsequent
chapters.
1.1 Background 9
There is also still a perception that 2DLC is not a mature method, that it is slow, the
data too complex and/or that that it cannot be used outside the specialist laboratory.
If this is your view, I hope perhaps this book will go some way to dispelling these
beliefs.
Before we go on to the more technical chapters of this book, I feel it is worth
stepping back to review the history of the technique. Those not so interested in this
aspect can skip forward to the next chapter if they wish.
As any student of chemistry knows, the father of liquid chromatography is
usually taken to be Russian botanist Mikhail Tsvet, who essentially developed
liquid-adsorption column chromatography in 1900 during his research on plant
pigments. Much has been written on Tsvet’s life and work (Abraham 2004; Ettre
1990; Livengood 2009; Sakodynskii 1981). The essentials are that he used calcium
carbonate as adsorbent and petrol ether/ethanol mixtures as eluent to separate chloro-
phylls and carotenoids (Ettre 1990). He first used the term “chromatography” in print
in 1906 and in 1907 he demonstrated his chromatograph for the German Botanical
Society, but the development of the technique stopped there for several decades.
Tsvet’s work was mostly ignored during his lifetime. Common factors cited for
this include the violent political upheaval in Russia at the beginning of the 20th
century, the fact that Tsvet originally published only in Russian (making his results
largely inaccessible to western scientists); and a paper by two prominent scientists of
the day who tried and failed to repeat Tsvet’s experiments and so denied the findings.
Later it was found that they did not follow his methods exactly and so used an overly
aggressive adsorbent (destroying the chlorophyll) and were not able to repeat his
results. This shows the importance of following published method in detail when
trying to recreate work. Tsvet’s method fell into relative (though not total) obscurity
(Ettre 1990). The technique was not fully revived until 10 years after his death. Today
of course, every student of chromatography knows the name Mikhail Tsvet.
The history of chromatography (while fascinating) does not explain the origin of
the term. Chromatography is a portmanteau of “chroma” from the Greek word for
“colour”, combined with “graphy”, meaning writing or recording-giving us ‘colour
writing’. Interestingly the word Tsvet in Russian can mean colour, which could give
us the alternative meaning of “Tsvet’s writing”. This makes more sense and is a nice
theory but, while certainly plausible there is no way to tell for sure if this is what was
intended. Tsvet could just as easily have picked the term because he was working
with plant pigments which were very colourful (see Fig. 1.6 for an example).
The issue is further complicated by the often-overlooked fact that the term chro-
matography was common well before 1900. Indeed, it was in use throughout the
19th century in connection with artists’ materials, especially colours and pigments.
George Field’s very famous and well-known book “Chromatography; or, a Treatise
on Colours and Pigments and of their Powers in Paint” was published in 1835 some
71 years before Tsvet used the term (Abraham 2004). Interestingly, Field does not
give the origin of the term either. This would indicate that either he was forgetful or
that the word chromatography was so well known in 1835 that he saw no reason to
spell out where it came from.
Fig. 1.6 Plant pigments extracted using column chromatography similar to that described by Tsvet
The full history is probably now lost but it does illustrate that the history of
science can be as fascinating as the science itself. Today the term chromatography
is not used in relation to art or art materials at all. It is thus interesting to note that
a term originally only used by artists is now almost exclusively used by scientists.
Although as my first lab manager said to me when I started my Ph.D.—“You have to
think of chromatography as an art as well as a science.” He did not mean it literally
but I have always found this to be good advice.
References
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Bushey MM, Jorgenson JW (1990) Automated instrumentation for comprehensive two-dimensional
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Callahan DL, Souza DD, Bacic A, Roessner U (2009) Profiling of polar metabolites in biolog-
ical extracts using diamond hydride-based aqueous normal phase chromatography. J Sep Sci
32(13):2273–2280
Creek DJ, Jankevics A, Breitling R, Watson DG, Barrett MP, Burgess KEV (2011) Toward global
metabolomics analysis with hydrophilic interaction liquid chromatography–mass spectrometry:
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da Silva RR, Dorrestein PC, Quinn RA (2015) Illuminating the dark matter in metabolomics. PNAS
112(41):12549–12550
Erni F, Frei RW (1978) Two-dimensional column liquid chromatographic technique for resolution
of complex mixtures. J Chromatogr A 149:561–569
Ettre LS (1990) Key moments in the evolution of liquid chromatography. J Chromatogr A 535:3–12
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distribution in multidimensional separation. J Chromatogr A 703(1–2):3–15
Guiochon G (2006) The limits of the separation power of unidimensional column liquid
chromatography. J Chromatogr A 1126(1–2):6–49
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biological systems. Metabolomics 14(8):101
Livengood J(2009) Why was M. S. Tswett’s chromatographic adsorption analysis rejected? Stud
Hist Philos Sci Part A 40(1):57–69
Pandohee J, Stevenson PG, Conlan XA, Zhou X-R, Jones OAH (2015) Off-line two-dimensional
liquid chromatography for metabolomics: an example using Agaricus bisporus mushrooms
exposed to UV irradiation. Metabolomics 11(4):939–951
Pesek JJ, Matyska MT (2012) A new approach to bioanalysis: aqueous normal-phase chromatog-
raphy with silica hydride stationary phases. Bioanalysis 4(7):845–853
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Chromatogr A 778(1):3–21
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Stevenson PG, Guiochon G (2013) Cumulative area of peaks in a multidimensional high
performance liquid chromatogram. J Chromatogr A 1308:79–85
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liquid chromatography through the use of high temperature ultra-fast gradient elution reversed-
phase liquid chromatography. J Chromatogr A 1122(1):123–137
Wishart S, Feunang YD, Marcu A, Guo AC, Liang K, Vazquez-Fresno R, Sajed T, Johnson D, Li
C, Karu N, Sayeeda Z, Lo E, Assempour N, Berjanskii M, Singhal S, Arndt D, Liang Y, Badran
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46(D1):D608–d617
Chapter 2
Basic Principles
Abstract Two-dimensional liquid chromatography is not simple but neither is it

as complex a technique as it is sometimes made out to be. New developments in
technology mean the technique is more and more accessible and user-friendly in terms
of method development, data analysis, and automation. While the extra dimension
increases separation power, peak resolution, and reproducibility it also increases the
complexity of the system. This chapter discusses some of the issues and constraints
that it pays to at least be aware of and hopefully understand before starting to design
2DLC methods. Common issues such as expected peak capacity, undersampling and
issues around columns and solvent compatibility are discussed. The point made that
it is important to keep in mind that the power of this technology is best demonstrated
when used to analyse samples containing hundreds or more components that cannot
be separated using a one-dimensional chromatographic system.
Keywords Analytical chemistry · Orthogonality · Peak capacity ·

Undersampling · Solvents · Surface coverage
2.1 Background
Since its introduction in the 1940s column chromatography has developed signif-
icantly into modern high-pressure liquid chromatography (hence the abbreviation
HPLC). As manufacturers of this form of instrumentation refined the technology,
they changed the name to high-performance liquid chromatography (which meant
no change in the abbreviation was necessary). In 2004 new technology allowed higher
pressure to be utilised and the term ultra-high pressure liquid chromatography, and
the new abbreviation, UHPLC, was introduced to the literature. This acronym was
later updated to ultra-high-performance liquid chromatography (again requiring no
change in abbreviation). Today much work done in this area is still considered to
be high-performance liquid chromatography, although UPLC is gaining ground in
many areas due to the fact it can make sample analysis a lot faster.
https://doi.org/10.1007/978-981-15-6190-0_2
14 2 Basic Principles
The “next frontier” is two-dimensional liquid chromatography where the extra

dimension increases separation power, peak resolution, and reproducibility. It also
increases the complexity of the system (and what might go wrong) and introduces
issues that are not considerations in standard HPLC. It pays to at least be aware of
some of these issues before jumping into 2DLC method development. Let us have a
look at some of them here.
2.2 Column Orthogonality
The fundamental issue of LC × LC is the combination of two different separation

mechanisms i.e. the combination of two columns which are different in both dimen-
sions and the nature of the stationary phase (and hence likely the mobile phase)
(Pandohee et al. 2015). Somewhat confusingly to some, chromatographers often
refer to the difference between phases in 2DLC in terms of the columns being orthog-
onal to each other. The word orthogonal comes from the Greek orthogōnios (“ortho”
meaning right and “gon” meaning angled). The term is used, in this case, to describe
events that are statistically independent, or do not affect one another in terms of
outcome.
Readers may also be familiar with the term orthogonal from applications such as
principle components analysis (PCA) in which the related term orthogonal projection
describes a method for drawing three-dimensional objects with linear perspective.
For those more used to speaking about orthogonality in terms of principle compo-
nents it is important to note that reference to orthogonal columns does not mean
that they are literally connected perpendicularly to each other but simply that the
column chemistries are diverse (and unrelated) (Pandohee et al. 2015). The greater
the difference in column chemistry comparted to the sample and each other the better
the separation (Giddings 1984). We will go into this in more depth in Chap. 3.
To ensure high-resolution separation the columns should be different phases. The
choice of the most appropriate column set to achieve the desired separation is difficult
since the user must consider the different flow geometries and chemical interactions
occurring in the two columns. Choosing the first and second columns is the most
crucial and challenging factor as this determines the separation capacity of the system.
The best result is achieved when two columns separate the compounds via different
mechanisms because retention of the compounds by the stationary phase in the first
dimension is different from the retention mechanism of the second dimension. Non-
orthogonal separation will lead to no separation over the separation space, the peaks
will be clustered and along a diagonal line from the bottom left corner to the upper
right corner. For example, Fig. 2.1 shows a 2D intensity plot that might result if
a sample was run on a two-dimensional system with two identical columns (two
C18 columns say) run in identical conditions. Note that this latter point can be quite
important. For example, a reverse phase (RP) × reverse phase 2DLC system run with
a significantly different pH in each column can have a high peak capacity since in
such a case the chemistry in each dimension is quite different (Gilar et al. 2005).
2.2 Column Orthogonality 15
Fig. 2.1 Stylized 2DLC

separation using columns
with identical phases and
conditions
We can see in Fig. 2.1 that the separation times are identical in each dimension and
so the separation is not improved by the second column. Ideally, we would make use
of all the possible separation space (the blue square). In actuality this does not usually
occur, and the separation tends to occur left and right from the central diagonal line
(red lines in Fig. 2.2) This is known as the spreading angle.
The wider the spreading angle the better the separation. We must remember
however that we are unlikely to ever make full use of the available separation space
(though we can get quite close). This is because while in an ideal separation science
world all the peaks would be evenly spaced out (and resolved) over the available
separation space, in real life the peaks are randomly distributed. This means that
some areas of the separation space may still have coeluting peaks while other parts
are unused. No separation mode in 2DLC is likely to offer complete orthogonality
of separation and the practical peak capacity of 2D separation systems is reliant
on a number of factors such as the number of collected fractions and the system
chemistry-mobile phase, column conditions (e.g. pH and temperature), column type
etc. Some of these factors are discussed below. An excellent discussion on opti-
mising separations in online comprehensive two-dimensional liquid chromatography
is given in Pirok et al. (2017).
Fig. 2.2 Stylised 2DLC

separation using columns
with different phases
showing the spreading angle
(black)
2.3 Peak Capacity and Undersampling
Having discussed above that it is not possible to use all the separation space it is
worth delving into how many peaks we can reasonably expect from a 2D method
before we cover method development in Chap. 3. This is so that we are not overly
optimistic early on (and then disappointed later). Indeed, one of the first questions
generally asked about 2DLC is “how many peaks can it resolve”? Here one might
be tempted to use what has become known as the “product rule” for calculating the
theoretical peak capacity.
The product rule states that under ideal conditions the total peak capacity (PCt ) of
a two-dimensional liquid chromatography system is the product of the peak capac-
ities of the first (PC1 ) and the second (PC2 ) dimensions. This can be written as the
following simple equation.
PCt = PC1 × PC2
This equation, while a useful starting point usually overestimates the true resolving
power as it underestimates the (often quite severe) effects of under-sampling and
surface coverage (fcoverage ). But what do we mean by these terms? Let’s start with
under-sampling? This is a very important factor in 2DLC and indeed nearly all areas
of analytical chemistry.
Modern analytical instrumentation usually provides us with digital data. This
means the readings are not continuous but discreate measurements separated by time.
A peak leaving a column, for example, is measured multiple times by the detector.
2.3 Peak Capacity and Undersampling 17
Fig. 2.3 Illustration of the effect of sampling frequency on peak shape
What we see as a continuous peak on the computer screen is in reality a series of

measurements joined together by the computer, even though the response they are
measuring is usually continuous. We also need to keep in mind that commercial
chromatography and mass spectrometry software may perform various data trans-
formations (such as data smoothing) in the background that we are unaware of. This
is one reason for the recent push towards open data standards such as mzTAB (Griss
et al. 2014). An example of the effect of sampling frequency on peak shape is shown
in Fig. 2.3. It can hopfully be seen that fast sampling across multiple coeluting peaks
is essential if they are to be adequality resolved.
As we learned in chapter one 2DLC works by taking aliquots of the effluent from
the first dimension and injecting them onto the second. We also learnt that the second
column can be thought of as a type of chemically sensitive detector. Other forms of
detector such as mass spectrometers or spectroscopic techniques cannot measure
continuously but must cycle through values of mass or wavelength, or wait for a
light source to reflect from the target. Similarly, sample aliquots cannot be moved
instantaneously from the first to the second dimension. If nothing else, no matter
how fast it is, the switching valve takes some amount of time to move between the
load and the inject positions.
This means we always lose some sample from the first dimension and thus the
“sampling rate” across the first-dimension separation has a potentially large influence
on the final peak capacity that is not accounted for in the product rule equation above.
There will always be “undersampling” of the first dimension and this necessitates
the application of a correction factor (Cf ) to account for it. This is often referenced
as the Greek letter Beta (β) in the 2DLC literature.
A modified equation may then be written as
PCt = (PC1 /Cf ) × PC2
The number of times one samples across a peak as it leaves the first dimension can
have a large effect on how that peak is seen in the final chromatogram. Two closely
eluting peaks could easily be plotted as one if there are not enough sampling points
across the peaks to identify when one ends and the next begins.
By now one might have realised that choosing the proper sampling time is very
important in practical 2DLC work.
One might next ask what correction factor must be used? This is a tricky question
to answer since there is a strong interaction between the sampling time and other
system parameters, such as the first-dimension gradient time and first dimension
peak width, and thus first-dimension peak capacity. So how does one determine peak
capacity in 2DLC? In many cases the product rule described above will be adequate
as the number of peaks in the sample will be far fewer than the maximum resolving
power available for the system.
2.4 Surface Coverage
Now let’s look at surface coverage or fcoverage . This can be defined as the fraction of the
available separation space that is occupied by peaks. Ideally this latter variable would,
of course, be as close to 100% as possible while still having every peak resolved from
each other. As we shall see this is rarely the case because while we would like peaks
to be evenly spaced out throughout a chromatogram in real life peaks are subject to
random distribution and so will overlap. This is the basis of statistical overlap theory
developed by Davis and Giddings (1983). This is a mathematically complex model
but, in short, it means that in many cases a lot of potential separation space is not
used. Davis and Giddings showed, for example, that, relative to the maximum peak
content for closely spaced speaks, a randomly distributed chromatogram will never
contain/seperate more than around 37% of its potential peaks; 2DLC was later shown
to be potentially worse in this regard (Davis 1991). Maximising the value for fcoverage
is thus usually one of the main aims in 2DLC method development.
If we modify our equation for total peak capacity to include the fcoverage term, we
get something like
PCt = (PC1 /Cf ) × PC2 × fcoverage
There is also a scan delay for non-chemical directors so we could, if we were

quite picky, include a correction factor for the PC2 as well. This could be written as
PCt = (PC1 × PC2 × fcoverage )/Cf

or PCt = (PC1 /Cf1 ) × (PC2 /Cf2 ) × fcoverage
However, any such Cf is likely to be minimal and not affect the overall results so
can likely be ignored.
For most 2DLC users, particularly those just starting out in the field, using the
product rule to estimate peak capacity will be satisfactory (as long as one bears in
2.4 Surface Coverage 19
mind the answers won’t be exact) as the number of peaks that need to be resolved
will be less than the separation space available. We will look at how to maximize the
available peak capacity in Chap. 3.
For those who wish to delve into this issue further Li et al. (2009) recently
published a very useful equation for estimating peak capacity in 2DLC. This incor-
porated a correction for under-sampling of the first dimension and explored this
issues in some mathematical depth. Although they only tested their equation on low
molecular weight compounds there is no reason to think that larger molecules would
show much difference. Their results showed that not only is the speed of the second
dimension separation important for reducing the overall analysis time (which is not
surprising since the faster the second dimension separation the faster one can sample
from the first dimension) but it also plays a vital role in determining the overall peak
capacity when the first dimension is under-sampled. A surprising subsidiary finding
was that for relatively short 2DLC separations (much less than a couple of hours) the
first-dimension peak capacity was far less important than is commonly believed. This
means that when first dimension gradient times are 30 min or less it does not pay to
use very small particles or take any other steps to improve the first-dimension peak
capacity above a value of about 50–100. Only in very slow (>2 h) 2DLC separations,
which are rare, does it make sense to improve the first-dimension peak capacity.
Their second important finding was that the dependence of the productivity of the
second-dimension separation (peak capacity per unit of gradient cycle time) dictates
the choice of the rate at which the first-dimension separation should be sampled
to maximize the total 2DLC peak capacity. Of greater practical consequence is the
finding that the optimum values of the second-dimension productivity occur at second
dimension gradient cycle times, in the order of 20 s, which are within reach of existing
instrumentation. Other workers such as Pirok et al. (2017) have shown that smaller
particles will improve peak capacity for fast separations but only up to a point. This
means that other factors, such as column chemistry and solvent composition are
better areas to focus on for method optimisation than using ever smaller particle
sizes in your column (with associated back pressure and friction problems).
2.5 Gradient Elution Chromatography
Most people who work with liquid chromatography will be familiar with the differ-
ence between isocratic and gradient elution so one could be forgiven for wondering
why I am discussing solvents here. The reason is that 2DLC introduces some
extra considerations that it is worth being aware of before moving onto method
development.
Let’s start by reminding ourselves of the differences between isocratic chromatog-
raphy and gradient elution chromatography as this is very important in 2DLC. In
isocratic conditions, the mobile phase composition is held constant during the sepa-
ration while in gradient elution chromatography the mobile phase strength is varied
from an initially weak eluent composition to a stronger one (usually via a simple
linear gradient). The major operational difference between isocratic and gradient
elution is therefore that in gradient elution chromatography both the instrument and
the column must be re-equilibrated to the initial eluent composition at the end of the
run (Stoll et al. 2006).
There are some other important differences. In isocratic elution the peaks are
relatively broad and the peak width increases with retention time. In gradient elution,
the peaks are narrow with almost equal peak widths. In isocratic chromatography,
the retention time of a particular compound is linear with its retention factor (e.g.
constant in the initial eluent composition) so the higher the retention factor the longer
the elution time, all other factors being equal. One cannot assign a fixed retention
factor value to a compound when gradient elution is used as this value changes during
elution (the retention factor decreases throughout the gradient run). We can, however,
calculate the average retention factor value for the whole of the separation. What this
boils down to is that in gradient work a much wider range of solute retention values
can be handled in a given separation time.
Retention time in gradient elution chromatography must also take into account
what is called the delay time to account for the time it takes the mobile phase composi-
tion changes to propagate through the system and get to the column inlet. Fortunately,
the impact of the gradient delay can be compensated for by deliberately starting the
solvent gradient before the sample injection is made (known as a delayed injection).
The concept of the gradient delay is extremely important in the second dimension of
2D-LC systems.
All of the above can have a large impact on how we do 2DLC.
2.6 Solvent Considerations
An easy way to lose efficiency from a HPLC separation is to pay little attention to
sample preparation, in particular the solvent(s) in which the analyte is dissolved. As
most undergrad chemists will know, the solvent within the sample vial need only
vary a little in strength compared to the mobile phase (at the initial composition for a
gradient separation) for peak broadening to occur. This can quickly start to erode the
baseline between closely resolved peaks. If the solvent strength mis-match is even
higher, significant band/peak distortion can occur.
In a 2DLC separation, small aliquots of a chromatographic separation (the first
dimension) are sequentially transferred to a different separation environment (the
second dimension). This adds an extra layer of complexity since, as we have already
established 2DLC works best with two columns of differing chemistries and thus
potentially differing mobile phases.
The use of two columns means that mobile phase compatibility is an important
issue to consider in 2DLC method. While the use of different mobile phases adds to
the change in selectivity when performing a separation on two orthogonal columns
(HILIC × RPLC or SEC × RPLC), it can also result in problems such as completely
immiscible solvents, which would make the coupling of the columns impossible. It
2.6 Solvent Considerations 21
is ideal if the mobile phase from the first column contains less organic phase than
the second column.
A shallow gradient program in the first dimension will often provide better
peak capacity, while both isocratic and gradient elution can be used in the second
dimension. The use of isocratic conditions in the second dimension avoids the need
for column re-equilibration at the end of the separation, which is highly desirable
for fast separations. However, gradient programs are more efficient in eluting
well-retained compounds such that the chances of wrap-arounds are minimised
(Lesins and Ruckenstein 1989). It is therefore often preferable to do gradient elution
chromatography in the second dimension even though on a fast time scale it is much
easier to do isocratic elution.
All of the above means that trying to complete a 2DLC separation using a gradient
in the first dimension is inherently challenging because the concentration of the
aliquot transferred between dimensions (i.e. the second-dimension injection solvent)
is continually increasing. The solvent strength miss-match can deform the peak just
as it would in normal HPLC and remove any ability to extract meaningful data from
a separation.
An innovative solution to this problem was developed by Stevenson et al. (2014),
who utilised a third pump into the 2D instrumental configuration. This allowed them
to introduce a “counter gradient” to offset the changing mobile phase produced by the
first dimension. They also found that the second-dimension injection solvent could
be artificially manipulated so that a consistent composition was sent to the second
dimension. The counter gradient has significance in RP × RP, but also HILIC × RP
separations, where the mobile phases are at the opposite ends of the solvent strength
spectrum. The authors also created a free-to-use interactive application that calculates
the required counter method for a single step gradient to produce the required transfer
solvent strength.
Unfortunately, the counter gradient approach also has its limitations. It was found
that the mobile phase concentration after the counter gradient was limited to approx-
imately 20% of the solvent component. Below that threshold several experimental
design issues arose, including very slow first dimension times; counter gradient flow
rates above instrument max; analytical scale sample loops of insufficient size; and
first dimension dilution leading a worse limit of detection (Stevenson et al. 2014).
There is also the not insignificant problem of the conceptual and instrumental
complexity of plumbing in a third solvent pump to the system and ensuring all
solvents are miscible and of equal viscosity at each stage of the separations. As
yet nobody has tried a counter gradient method with reverse and normal phases
of chromatography, but it could be anticipated that the inclusion of a solvent that is
miscible in both of these mobile phase environments will improve their compatibility.
This could dramatically enhance the time and space efficiency of 2D-HPLC analyses.
2.7 Column Temperature
In gas chromatography separations the temperature program used is an essential

part of a successful separation. The use of elevated temperature in HPLC has a
similar theoretical promise as using smaller particles with UHPLC-namely better
performance and reduced analysis time. If the column temperature is increased, the
chromatographic separation process becomes faster and, in general, more efficient.
Both lower mobile phase viscosity and increased temperature improve diffusion
during the chromatographic process. However, the percentage decrease in retention
time is usually not the same for all compounds of a sample mixture and changes
in peak spacing are common. Aqueous solvents are known to exhibit less polar
characteristics as they are heated and therefore if used under isocratic conditions,
temperature programming can provide the benefits of gradient chromatography in
manipulating retention, whilst maintaining a constant mobile phase eluent.
Temperature programming is a potentially valuable tool for 2DLC and suited for
applications that demand fast analysis times and high peak capacity as the deleterious
effects of solvent mismatch and reduced sensitivity from other complex interfacing
solutions are avoided. Two columns with different chemistries can be housed with
independent temperature control for each column, providing versatility for orthog-
onal separations. Just as in HPLC in general, there has so far been less focus on
temperature primarily because metal LC columns and the separation beds are slow
to heat (and cool) and difficult to heat uniformly and separations can be effectively
achieved using other methods.
The above notwithstanding Holland et al. (2016) used temperature programming
to manipulate solvent elution strength in place of a mobile phase concentration
gradient. This ensured that all eluent fractions transferred into the second dimension
were of identical solvent composition, i.e. the second-dimension injection solvent did
not increase during the analysis. When applied to a complex natural product extract
of coffee, the separation was completed in 35 min and had an orthogonality value
of 35% and a spreading angle of 52°. The use of temperature programming avoided
solvent incompatibility at the separation interface, ensuring peaks in the second
dimension retained symmetrical profiles which led to improved peak capacity and
reduced retention times.
At first glance temperature programming seems promising, so why is it not more
widespread? The need for samples and HPLC columns that are stable at high temper-
atures is limiting for many applications. The extended thermal re-equilibration times,
in the order of 40 min, are also unfavourable for high throughput routine analysis,
requiring further attention from the instrumental and column design viewpoint.
2.8 How to Use 2DLC 23
2.8 How to Use 2DLC
A question that naturally arises is; what kinds of problems are best addressed using
2DLC? The simple answer is complex ones. This can be seen in the literature where
the vast majority of applications deal with very complex naturally occurring mixtures
(biological cells, blood, urine, environmental samples and so forth). It is also worth
keeping in mind the adage that one does not take the Ferrari to do the supermarket
shopping (unless you want to show off of course). The real power in 2DLC is in
being able to resolve complex mixtures quickly. Indeed today, what once took 6 h
can take as little as 30 min.
If you have fewer than 150–200 peaks to resolve in a reasonable timeframe
of around 30 min you are likely better sticking with HPLC (with UHPLC if
between 300–500) (Poppe 1997). Today’s samples, however, increasingly call for
the separation of thousands of componants and so 2DLC could have use in a wide
range of areas. The increased separation power of two-dimensional chromatography
is of great potential in a large variety or areas, particularly for compounds that
are too sensitive for mass spectrometry but contained in matrices too complex for
standard LC analysis. The combinations of new column chemistries that could be
used are many and varied and the use of size exclusion and chiral columns in the
first dimension offers up many further opportunities for novel applications.
References
Davis JM (1991) Statistical theory of spot overlap in two-dimensional separations. Anal Chem
63(19):2141–2152
Davis JM, Giddings JC (1983) Statistical theory of component overlap in multicomponent
chromatograms. Anal Chem 55(3):418–424
Giddings JC (1984) Two-dimensional separations: concept and promise. Anal Chem 56(12):1258A–
1270A
Gilar M, Olivova P, Daly AE, Gebler JC (2005) Orthogonality of separation in two-dimensional
liquid chromatography. Anal Chem 77(19):6426–6434
Griss J, Jones AR, Sachsenberg T, Walzer M, Gatto L, Hartler J, Thallinger GG, Salek RM, Steinbeck
C, Neuhauser N, Cox J, Neumann S, Fan J, Reisinger F, Xu Q-W, del Toro N, Pérez-Riverol Y,
Ghali F, Bandeira N, Xenarios I, Kohlbacher O, Vizcaíno JA, Hermjakob H (2014) The mzTab
data exchange format: communicating mass-spectrometry-based proteomics and metabolomics
experimental results to a wider audience. Mol Cell Proteomics 13(10):2765–2775
Holland BJ, Conlan XA, Francis PS, Barnett NW, Stevenson PG (2016) Overcoming solvent
mismatch limitations in 2D-HPLC with temperature programming of isocratic mobile phases.
Anal Methods 8(6):1293–1298
Lesins V, Ruckenstein E (1989) Gradient flow programming: a coupling of gradient elution and
flow programming. J Chromatogr A 467:1–14
Li X, Stoll DR, Carr PW (2009) Equation for peak capacity estimation in two-dimensional liquid
chromatography. Anal Chem 81(2):845–850
Pandohee J, Holland BJ, Li B, Tsuzuki T, Stevenson PG, Barnett NW, Pearson JR, Jones OA, Conlan
XA (2015) Screening of cannabinoids in industrial-grade hemp using two-dimensional liquid
chromatography coupled with acidic potassium permanganate chemiluminescence detection. J
Sep Sci 38(12):2024–2032
Pirok BWJ, Gargano AFG, Schoenmakers PJ (2017) Optimizing separations in online comprehen-
sive two-dimensional liquid chromatography. J Sep Sci 41(1):68–98
Stevenson PG, Bassanese DN, Conlan XA, Barnett NW (2014) Improving peak shapes with
counter gradients in two-dimensional high performance liquid chromatography. J Chromatogr
A 1337:147–154
Stoll DR, Cohen JD, Carr PW (2006) Fast, comprehensive online two-dimensional high performance
liquid chromatography through the use of high temperature ultra-fast gradient elution reversed-
phase liquid chromatography. J Chromatogr A 1122(1):123–137
Chapter 3
Method Development
Abstract Two-dimensional liquid chromatography offers a way to greatly increase

the number of compounds that can be separated, detected, and quantified in a single
analytical run to a greater degree of sensitivity and selectivity than standard HPLC.
Many potential users of two-dimensional technology are put off by its apparent
complexity, but modern 2DLC today is greatly facilitated by advances in software
that makes the technology relatively easy to run. The high-pressure capability of
modern instrumentation allows high flow rates and correspondingly high-speed anal-
ysis and one no longer has to count individual peaks by hand. Multidimensional
chromatography offers a potential solution to many separation problems but method
development in 2DLC is more complex that standard HPLC and users must consider
factors such as the need for two independent retention mechanisms (column orthog-
onality), solvent mismatch, and back-mixing (sample wraparound) while ensuring
robust analytical performance from increased instrumental complexity. Some basic
considerations in method development for 2DLC are also discussed here.
Keywords Columns · Computability · Modulation · Offline · Online · Separation

mechanisms
3.1 Background
Most analytical chemists reading this will have had to develop and apply new
methods, probably for a range of instruments, as part of their everyday job. To do so
they would consider the sample type and then think of the best column chemistries to
use. At the end of the day, once you understand the principles of method development
it can be repeated many times over. Now, since, as we have discussed in Chaps. 1–3,
an LC × LC method is “simply” the product of two 1D separations should not method
development be similarly within reach of most chromatographers? The answer is not
as simple as one might think.
Before we go much further I should make readers aware that there is an excellent
technical resource in the form of a searchable 2DLC method database available. A
collaboration between two leaders in the field, Dwight Stoll of Gustavus Adolphus
https://doi.org/10.1007/978-981-15-6190-0_3
26 3 Method Development
College and Bob Pirok of the University of Amsterdam it can be accessed at www.mul
tidlc.org and is a comprehensive source of information on multi-dimension separa-
tions. To make sense of all the material on this site however once needs to understand
some of the important factors in LC × LC method development. Understanding the
principles is also of use if one is trying to develop a totally new method for a new
sample types.
3.2 What Factors Should I Consider?
The important factors in 2DLC are just the same as in standard LC. The choice
of column packing material, particle size, flow rate and mobile phase(s) employed
are critical factors in driving the separation that must be optimised to obtain the
best result. When performing two-dimensional (2D) chromatography two separate
separations need to be optimised simultaneously. We also need to keep in mind that
there are two main forms of 2DLC (comprehensive and heart cutting) as well as two
main types of method development (offline and online).
The use of two columns and thus multiple mobile phase setups introduces extra
complications that the analyst must consider. These include fundamental factors
such as column selection, sampling rate, solvent compatibility, second dimension
analysis time, and sample loop volumes among others. All need to be considered
and optimised for a successful analysis (Schoenmakers et al. 2006). This makes
the development of a two-dimensional liquid chromatography method a little more
challenging. So, what are the main issues when developing a method that uses the
second dimension? Before we jump into detailed discussion let’s look at some of the
more common ones. They include the following.
• Means to transfer the peak from first dimension to second dimension. Some form
of modulator/transfer valve is usually used for this. System pressure differences
are likely to be an issue if transferring an analyte from the first dimension to the
second dimension directly.
• If using a sample loop, the loop size, transfer volume, and percentage filling (of
the loop) all need to be considered.
• If using a trap column (which we will discuss later), consideration of the column
chemistry is critical.
• Mobile phase compatibility and associated solvent effects between both dimen-
sions are a common issue and if the two methods contain incompatible mobile
phases (such as ion exchange (IEX) × RP), the compatibility of high salt content
with organic solvent (s) needs to be considered.
• Mobile phase compatibility with any associated detector is also a concern. For
example, if MS is the downstream characterization then MS unfriendly mobile
phases need to be removed before the effluent reaches the MS detector.
We should also perhaps consider the differences between offline and online
(automated) method development.
3.3 Offline Method Development 27
3.3 Offline Method Development
Offline 2DLC has actually been around for around 40 years but has recently under-
gone something of a resurgence. It is simpler than online 2DLC and has been applied
in studies on food, (Dugo et al. 2004) traditional medicines, (Ma et al. 2006) peptide
digests, (Marchetti et al. 2008) metabolomics and lipidomics (Li et al. 2014) amongst
others. It does not require complicated instrumental set-up and can be performed with
a standard 1DLC system consisting of an autosampler, a pump (usually binary or
quaternary), a column oven, a detector, and a fraction collector to sample the first
dimension. The sample is injected and the eluent from the first column is collected
in sample vials using the fraction collector. These fractions are then re-analysed via
the second column.
Since the two dimensions are independent there is no restraint on the analysis
time of the second dimension. This implies that longer columns that will provide
more theoretical plates can be used in both the first and second dimensions. Hence,
the offline mode often affords a greater total peak capacity. Moreover, the volume
of eluent being transferred can be increased accordingly with the volume of the
second-dimension format to improve the sensitivity.
Fraction collection also allows manipulation of the sample, such as pre-
concentration, derivatisation, and/or reconstitution into the appropriate mobile phase,
prior to injection into the second dimension (Fairchild et al. 2009a). Although the
instrumental set-up for offline 2DLC mode is simpler, it is also more labour inten-
sive and since the eluent is in contact with more tubes there is a higher risk of
cross-contamination, sample loss and the introduction of artefacts. The total analysis
time of an offline analysis is significantly higher (a few hours or days) than an online
analysis (Fairchild et al. 2009a).
Protocols for offline 2DLC method development have been outlined previously
by Horváth et al. (2009). Important factors to note are that the first and second
dimension conditions (column type, mobile phase composition, flow rate, gradient
program and temperature) must usually be optimised separately. The choice of which
two columns to use for a given separation is usually obtained via selectivity studies
using multiple column types in conjunction with knowledge of column chemistry and
this was recently the subject of a detailed review by Bassanese et al. (2015). A good
example of this in action can be seen in Pandohee et al. (2015). The final separation
can be maximized by optimising solvent composition, flow rate and temperature,
for example, just as in standard LC. A sampling rate is then determined to maintain
separation efficiency and avoid undersampling. For a truly comprehensive separation
at least 3 or 4 fractions per peak should be analysed (Bassanese et al. 2015).
3.4 Online Method Development
An online 2DLC method can be complicated to develop and involves compromises

in terms of volume of effluent transferred, analysis time of the second dimension and
solvent compatibility (Fairchild et al. 2009b). Schoenmakers et al. (2006) proposed a
protocol for online 2DLC method development covering suitable column dimensions
(length and diameters), particle sizes, flow rates, and second-dimension injection
volumes (i.e. loop sizes). The protocol suggests that the experimenter selects the first
dimension parameter details, which can include the maximum workable pressure
drop, the smallest column diameter, and the maximum acceptable (or essentially
the total) analysis time, per sample (Schoenmakers et al. 2006). Poppe plots (Poppe
1997), which offer a clear and unambiguous way to discuss the performance limits of
separation systems, are then constructed for these columns and a compromise is made
for the best conditions to factors such as particle size, flow-resistance factor, eluent
viscosity and analyte diffusivity (Schoenmakers et al. 2006). These are then used to
determine the second-dimension parameters including particle diameter, number of
plates, column length, and peak capacity.
One of the main reasons for the increase in complexity for online 2DLC is that
the speed of the second separation must be considerably higher compared to the first
dimension. Ideally, the sample should be clear of the second dimension prior to the
next injection otherwise this results in additional stress to the secondary pumps, as
they are working at higher pressures. A further disadvantage is the increased price
compared to single dimension systems.
For the successful development of an online LC × LC method, it is again important
that the two dimensions are orthogonal to allow good use of the two-dimensional
separation space. Gilar et al. (2005) investigated the selectivity of various modes such
as RP, SCX (Strong Cation Exchange), SEC (Size Exclusion Chromatography), and
HILIC to identify LC × LC setups with useful orthogonality. It was found that an
RP phenyl column × RP C18 combination, both working at a pH of 2.6 would be a
correlated system, while the use of a RP C18 × RP C18 at a pH of 2.6 and 10 in the
first and second dimension, respectively, would use a bigger portion of the separation
space. Moreover, combining a RP C18 × HILIC or RP C18 × SCX would provide
orthogonal separations that were especially good for peptides.
The analysis time of the second dimension is the next parameter to optimise.
Since the speed of the second separation dictates the sampling rate and flow rate
of the first dimension, short and rapid second dimension separations allow a higher
number of fractions to be transferred to the second dimension (thus maintaining peak
capacity) as well as appropriate sampling of the first dimension (remember this is
3–4 fractions per peak). Numerous strategies, such as the use of monolithic columns,
high temperature chromatography with zirconia columns and/or the use of sub 2 µm
particles have been developed to increase the second analysis speed. The latter can
be highly advantageous as the use of small particles provides more theoretical plates
and hence an increase in resolution in the second dimension. The use of core-shell
particles is also very attractive for instruments that do not possess a UPLC pump as
3.4 Online Method Development 29
their second pump as they operate at lower backpressure than conventional particle
stationary phases but still provide high-resolution separations.
The second-dimension formats used are usually short (30–50 mm in length) and
are conventional bore columns (internal diameter of 4.6 mm) to provide the shortest
analysis time possible and allow the introduction of relatively large fraction volumes
from the first dimension. The first-dimension flow rate can be then adjusted (usually
in the order of nano or micro litres per minute) so as the peaks are being sampled
3–4 times. In doing so, the resolution obtained in the first dimension is maintained
throughout the second dimension. Murphy et al. (1998) showed that if the first dimen-
sion is under-sampled, that is the modulation ratio is less than 4, then some of the
first dimension is lost in the sample loop. It has also been observed that at a modu-
lation ratio of 3 the loss in resolution did not exceed 10% (Davis et al. 2007).The
use of narrow bore (i.d. 1.0 mm or 2.1 mm) or capillary columns is therefore highly
recommended. In the online mode, the optimum resolution is mainly attributed to the
first dimension, with the second dimension providing the change in selectivity. To
maximise the efficiency, long columns or serially coupled columns, are often used.
Mobile phase compatibility in the two dimensions is an important issue to consider
when developing a 2DLC method. While the use of different mobile phases adds to
the change in selectivity when performing a separation on 2 orthogonal columns
(HILIC × RP or SE × RP), it can also result in completely immiscible solvents,
which would make the coupling of the columns impossible. It is therefore ideal that
the mobile phase from the first column contains less organic phase compared to
the second column. In the first dimension, a shallow gradient program should be
used because this will provide better peak capacity, while both isocratic and gradient
elution are used in the second dimension. The use of isocratic conditions in the second
dimension avoids the need for column re-equilibration at the end of the separation,
which is desirable for fast separations. However, gradient programs are more efficient
in eluting well-retained compounds (Lesins and Ruckenstein 1989).
A 2DLC can be coupled with all LC detectors including ultra-violet, photo-diode
array, mass spectrometer, or evaporative light scattering detection. It is important
to note that a high acquisition rate is needed to ensure that enough data points are
being collected from the sharp peaks obtained from the high-speed second analysis.
Moreover, the use of a mass spectrometer, which is orthogonal to LC separations
(since it separates by mass to charge ratio), offers additional information on the
sample components. Therefore, when co-eluting compounds from highly complex
samples they can be identified according to their mass. A summary of the different
forms of liquid chromatography is given in Table 3.1.
Of course, an HPLC column also needs a mobile phase if it is to be of any use
and these bring their own challenges.
Table 3.1 Summary of advantages and disadvantages of offline and online two-dimensional liquid
chromatography
Offline Online
Interface Fraction collector Valve
Sample Yes, including pre-concentration Not always possible
treatment/pre-concentration and derivatisation
before second dimension
injection
Total analysis time Significantly high (from hours Relatively fast (almost as
to days) long as a one-dimensional
separation)
Analysis time of second No time limit on separation time Second dimension analysis
dimension of second dimension time has to be less than first
dimension analysis time so
that the next sample can be
injected
Peak capacity Very high peak capacity can be High
obtained by using two long
columns
Wrap around None Will occur if second
dimension is not finished with
all compounds eluted before
the next injection
Automation Not automated, risk of Automated
contamination, labour intensive
Data representation Stacked or contour plots 2D (contour or projection)
and/or 3D
3.5 Column Selection (Orthogonality)
One critical point that is worth spending some time on is column orthogonality. The
fundamental issue of LC × LC is the combination of two different separation mech-
anisms i.e. the combination of two columns which are different in both dimensions
and the nature of the stationary phase (and hence likely the mobile phase). We also
need to keep in mind the fact that the second column is usually shorter than the first.
This can be seen in the 2DLC set up in Fig. 3.1.
The selection of two orthogonal columns for LC × LC separation can be a labour
intensive and time-consuming process and in many cases is an entirely trial-and-error
approach. Knowledge of the column and sample chemistries involved can help speed
up the process of conducting a selectivity study comparing multiple columns to see
which two produces the most different results with the same sample and then using
these two columns as the two different dimensions.
Pandohee et al. (2015) for example tested six different stationary phases (Luna
Cyano (CN), Luna C18, Luna NH2 , Luna pentafluorophenyl (PFP), Luna Phenyl-
hexyl and a Kinetex C18. All columns had dimensions of 150 mm by 4.6 mm, a
3.5 Column Selection (Orthogonality) 31
Fig. 3.1 Agilent Technologies 2D-LC system showing the two columns. The primary (C-18) on
the left of the picture and the shorter secondary (phenyl-hexyl) column on the top right have been
highlighted in red and blue boxes respectively
particle diameter of 5 µm and a pore size of 100 Å. The selectivity study involved
comparing the previously listed columns using a mobile phase composition that
started at 5% methanol (95% water) and rose to 100% methanol by 20 min; the
methanol was held at 100% for another 10 min to ensure that all components were
eluted from the column.
The quality of the separation was determined by comparing the number of peaks
and the use of the total separation space for each column. The same procedure
was repeated for all columns that could potentially be used as a second dimension
and the two columns providing the most dissimilar separations (the CN and the
C18) were chosen for the first and second dimensions respectively. Both dimensions
were then further optimised (solvent composition, flow rate, temperature) in the
laboratory so that the analysis time was as short as possible. The differences in the
1DLC chromatograms obtained for each column (with all other variables being kept
identical) are shown in Fig. 3.2.
An alternative approach is to use in silico testing to predict retention times on
different columns. For example Burns et al. (2016) showed that in silico optimisation
in the form of a data processing pipeline, created in the open-source application
OpenMS, could be developed to map the components within the mixture of equal
mass across a library of HPLC columns. LC × LC separation space utilisation was
compared by measuring the surface coverage. This allowed for significantly quicker
2DLC phase selection and the predicted separation space utilisation closely matched
actual results.
Fig. 3.2 Selectivity study of 4 stationary phases A: CN, B: NH2 , C: PFP, D: Phenyl-Hexyl and
E: C18. All columns were Luna columns of 150 mm × 4.6 mm × 5 µm. The solvent protocol
started from 5% methanol to 100% by 20 min, the organic phase was held at 100% for 10 min (after
Pandohee et al. 2015)
A recent development in this area is the use of the commercial software to predict
compound retention times as well as the effects of varying multiple parameters
such as pH, temperature, and buffer concentration. This software allows the choice
of columns and solvents etc. to be optimised in silico before running experiments
(Andrighetto et al. 2014). As such, in silico testing has clear, financial, temporal and
environmental benefits.
A recent review of 2DLC looked at which column combinations were most
common for both comprehensive 2DLC and heart cutting 2DLC (Pirok et al. 2019).
For the former, RPLC × RPLC was the set up in ~35% of published papers. This
was followed by HILIC × RPLC (~15%), IEX × RPLC (~5%) and SEC × RPLC
(~4%). In LC-LC the order was different. RPLC-RPLC was again in the lead (~36%
of published papers) but second was SEC-RPLC (~11%), then lastly IEX-RPLC
(~7%) and HILIC-RPLC (also ~7%).
3.6 Solvents 33
3.6 Solvents
In gas chromatography (GC) the mobile phase is specifically chosen to be inert to

the solutes and has no role in relative retention or selectivity of the analytes (solute-
mobile phase interactions are near zero) and a solute’s vapour pressure controls
distribution. With LC, solute-mobile phase interactions are significant and solute
distribution into the mobile phase is increased well above what would be provided
by the solute vapour pressure alone.
The 1DLC separations that are to be combined into an LC × LC method must be
carefully matched in terms of selectivity, compatibility, orthogonality, and resolution.
A good review of this topic (with some very useful tables and diagrams) is given in
Pirok et al. (2017).
Understanding solvent chemistry is also vital in 2DLC. Using two columns comes
with an additional issue around solvent mismatch (for example if one wanted to
combine normal and reverse and phase columns). The use of ancillary solvents such
as n-butanol can be used to reduce solvent mismatch and column re-equilibration
times. It is also vital that solvents that are carried between the columns do not have
different viscosities or miscibility or there may be issues with viscous fingering or
flow patterns. Viscous fingering (also known as Saffman–Taylor instability) is the
unstable displacement of a more viscous fluid by a less viscous fluid. It can occur
even in the absence of porous media. In terms of HPLC, it is a phenomenon discussed
extensively by Shalliker et al. (2007).
Peak separation can be improved in 2DLC using variable second-dimension gradi-
ents. These were discussed a little in chapter two. Let’s look at the effect they can
have on a separation. Figure 3.3 below shows four different reverse phase 2DLC
elution programs.
In each case the solvents were 0.1% formic acid in water (solvent A) and 0.1%
formic acid in acetonitrile (solvent B) for the first dimension, and 0.1% formic acid
Fig. 3.3 Solvent composition of four different reverse phase 2DLC elution programs
Fig. 3.4 Identical sample mix run through the four different solvent composition programs in
Fig. 3.3
in water (A) and 0.1% formic acid in methanol (B) for the second dimension. The
red line in each graph refers to the constant increase in % of solvent B of the first
dimension over the run. The blue line refers to the %B of the second dimension. We
can see that in each case this rises to a certain percentage and then returns to 5%
before the next injection, except for method 4 where it gradually increases with each
new injection.
If we take these methods and use them to run a test mix containing a
range of simple, yet closely related compounds, namely uracil, sulfamethazine, 2-
hydroxyquinoline, phenol, acetanilide, methyl paraben, acetophenone, ethyl paraben,
propiophenone, propyl paraben, N-N-diethyl-m-toluamide, butyrophenone, butyl
paraben, toluene benzophenone, valerophenone, heptyl paraben what will the results
be? This is shown in Fig. 3.4.
As can be seen that while there is a lot of similarities, method two makes the
best use of the separation space. Optimising even this relatively simple separation
took a several days. As a result, it is possible to spend more than twice as long to
develop an acceptable LC × LC method than to develop two 1D LC methods. This
can dramatically increase the method development costs of LC × LC relative to 1D
LC and other analytical techniques, which can be a high barrier if one is faced with an
urgent analytical question. However, there have been a number of advances in 2DLC
particularly around modulation and method optimisation software which have made
this task much easier.
3.7 Modulation 35
3.7 Modulation
The modulation (or switching) valve is the heart of the (online) 2DLC system. In stan-
dard 2DLC the modulation is passive-fractions from the first dimension are shifted
onto the second. It has been proposed that modulation periods are most favourable
when they are adjusted to be ∼2.2−4 times the standard deviation of a first dimen-
sion peak in order to avoid the need for excessively short run times in the second
dimension (Horie et al. 2007). But there is far more to modulation than timing.
Recently several researchers have started to introduce forms of active modulation
in which some modification of the effluent from the first dimension is undertaken
before it is moved on. Such systems include Stationary Phase Assisted Modulation
(SPAM), Active Solvent Modulation (ASM) and Vacuum Evaporation modulation
(VEM). An excellent review of fundamental improvement in the practical applica-
tions of 2DLC with active modulation (and other factors) is given in Pirok et al.
(2019) but we will discuss the topic briefly here.
In SPAM, rather than using large storage loops, analytes are effectively filtered
out of the first dimension effluent using low-volume trapping columns which can trap
and concentrate the analytes (Baglai et al. 2018; Gargano et al. 2016). Optionally,
the first column effluent may be diluted using a weak eluent to facilitate retention on
the traps. Although the system relies on compound retention in the traps it results in
a reduction of analysis time and removal of incompatible fractions. The modulation
volume is now no longer a limiting factor due to the trap column.
Developed for RP in the second dimension, ASM sends part of the second dimen-
sion flow back, via a bypass capillary, to act as a diluent for the first dimension effluent
fraction injected into the second column (Stoll et al. 2017). This method is robust
and significantly reduces breakthrough effects and enables on column focussing of
the injected analyte. Modulation volume is still a limiting factor in this approach.
In VEM the incompatible solvent is removed from the first-dimension effluent via
evaporation (which can be vacuum-assisted). This method is highly experimental at
the time of writing (April 2020). It has only been demonstrated for combinations of
NPLC and RPLC to date and analyte loss is a concern, although this can be addressed
with the use of a suitable membrane. Some analytes may also only redissolve slowly
(if at all) once one solvent is evaporated (Groeneveld et al. 2019). It should also be
noted that all modulators increase the complexity of the already challenging method
development for 2DLC. A lot of research work is currently being undertaken to try
and solve this problem, including extensive use of computer-aided development.
Some readers may be wondering if they can use a guard column (if they are using
one) as a trap column? Here it pays to remember that trap columns and guard columns
are different. A guard column generally has the same ID particle size and material
and the same frit size as the analytical column. It is used to protect the analytical
column.
A trap column is typically packed with a different packing material than the
analytical column. The range of packing materials vary and could include RP, IEX
and mixed-mode stationary phases with particle sizes from 10 to 30 microns. Trap
columns can also have a larger frit size to allow higher flow rates during the loading
process, up to 5 mL/min, while keeping the back pressure within the optimum range
of the pumps.
It is generally advisable not to use a guard column as a trap column, primarily
because of its variable particle size packing. A guard column could result in high
back pressure at higher flow rates during the loading step. The mass of the guard
column would also most likely not be sufficient for effective trapping.
Compared with directly transferring an analyte from a first dimension to a second
dimension column, a trap column reduces system pressure at transfer due to larger
particle size and shorter length than a typical LC column. The main use of a trap
column is to retain the analyte in a range of volumes to facilitate quantification in
the second dimension. Along with at/on-column dilution, it can effectively focus the
analyte and prevent break-through. A sample loop thus won’t have the same functions
as a trap column.
If one thinks about it, the technology platform that has been developed for 2DLC
also creates a number of other possibilities. All kinds of physical (e.g. dissolution) or
chemical (e.g. enzymatic or light-induced degradation or detection) processes can be
made to take place between the two separation stages in 2DLC. This allows for a wide
variety of experiments to be performed within a single, efficient, automated analysis
(Groeneveld et al. 2019). This may lead to many novel experimental approaches in
future, and it will be exciting to see where this goes.
3.8 Conclusion
The complications that can be a concern in 2DLC method development are gradu-
ally being removed. Detector-sensitivity and phase-system compatibility issues can
largely be solved by using active-modulation strategies. A large number of applica-
tions are published in the literature and robust instruments are commercially avail-
able. The recent developments in modulation technology have unlocked new possi-
bilities in hyphenation of 2DLC with mass spectrometry and solvent incompatibility
is often no longer an issue. Similarly to its big brother GC × GC, the advance in
computer processing power has facilitated automated method optimization to the
end that 2DLC has matured well enough for routine applications in analytical labs
and there has been great activity in the development and application of 2D-LC tech-
niques. Both heart-cut (LC-LC) and comprehensive (LC × LC) techniques appear
to have a bright future.
References 37
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two-dimensional high performance liquid chromatography for differentiation of ephedrine and
pseudoephedrine based methamphetamine samples. Forensic Sci Int 244(1):302–305
Baglai A, Blokland MH, Mol HGJ, Gargano AFG, van der Wal S, Schoenmakers PJ (2018)
Enhancing detectability of anabolic-steroid residues in bovine urine by actively modulated online
comprehensive two-dimensional liquid chromatography–high-resolution mass spectrometry.
Anal Chim Acta 1013:87–97
Bassanese DN, Holland BJ, Conlan XA, Francis PS, Barnett NW, Stevenson PG (2015) Proto-
cols for finding the most orthogonal dimensions for two-dimensional high performance liquid
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Burns NK, Andrighetto LM, Conlan XA, Purcell SD, Barnett NW, Denning J, Francis PS,
Stevenson PG (2016) Blind column selection protocol for two-dimensional high performance
liquid chromatography. Talanta 154:85–91
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capacity in comprehensive two-dimensional separations. Anal Chem 80(2):461–473
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Chapter 4
Data Analysis
Abstract 2DLC can resolve far more peaks than standard HPLC but it also produces
far more complex data. We are at a point in analytical science where running samples
and generating terabytes of data is often the simplest part of the workflow and
analysing the data properly takes up the majority of the analyst’s time. The ques-
tion then becomes how to draw meaningful insights from these datasets, and once the
data’s salient features are extracted, how can we best identify them, and infer meaning
from that list of identified compounds. The data must be processed properly to create
useful chromatograms, identify all the peaks, and generate new knowledge from the
data obtained. There are a number of different ways to do this ranging from pre-
process smoothing algorithms to geometric approach factor analysis. Understanding
these methods as well as the format of the raw data is an important part of the 2DLC
workflow.
Keywords Algorithm · Chemometrics · Deconvolution · In silico · Processing ·

Software
4.1 Background
It will come as no surprise to learn that that the strengthening of the separation power,
with peaks coming from two dimensions of separations, leads to the formation of a
correspondingly more crowded data plot. It is perhaps also not surprising therefore
that LC × LC files are much bigger than their one-dimensional counterparts. Indeed,
if we imagine an entire profile of a complex sample, it is easy to understand that the
amount of data coming from the 1D separation, once expanded to the orthogonal
plane, becomes very large. Each modulation produces continuous short, fast and
practically isocratic 2D chromatograms, leading to final sizes of 500 Mb or more.
File sizes are also strictly linked to acquisition frequency.
The substantial number of large data files produced by 2DLC require advanced
software for data processing, graphing and interpretation (Guiochon et al. 2008).
Dealing with two dimensions of separation requires a large amount of processing
power to enable visualisation and analysis (both quantitative and quantitative). This
https://doi.org/10.1007/978-981-15-6190-0_4
40 4 Data Analysis
introduces two problems to be solved. One, how to collect and save the data files and
two, how to easily handle and process the acquired data.
Sample pre-treatment and pre-separation are used to reduce the complexity of the
sample in conjunction with deconvolution software; however these are not always
successful (Nikitas et al. 2001; Vivó-Truyols et al. 2002). They can increase analysis
time, sometimes introduce artefacts into the data and may not allow a good represen-
tation of the entire sample. 2DLC has therefore also been reliant on the development
of new software capable of meeting the requirements of the modern analyst.
In the early days of 2DLC the general trend was to develop home-made software
via programming languages such as such as Mathematica and MATLAB or freeware
such as R or Python. One of the simplest ways to do the work in such software is to
quantity all the separated spots present in the bi-dimensional plane via the summation
of all the single peak areas that could be linked to the same constituent. This is time-
consuming and requires advanced programming knowledge but usually gives good
results.
Today, commercial 2DLC systems come with very powerful software that auto-
matically generates the required chromatograms and data tables. However, while
perhaps not a concern for most analysts. The fact is that most commercial programs
are proprietary. One can’t see what they are doing with the data or how they are
recording it in the first place. Is a signal smoothing algorithm applied to the raw
signal from the detector before the data is plotted? In many cases, we simply don’t
know. It is worthwhile therefore having some discussion about data analysis in 2DLC.
This chapter aims to give the reader a general overview of the area and its impor-
tance. Readers that wish to dive into the applications of chemometrics in one- and
two-dimensional chromatography in more depth are directed to the excellent recent
review of the subject by Bos et al. (2020).
4.2 Displaying the Data
Because they contain a retention time and a detector measurement for each dimension
2DLC chromatograms are generally displayed as contour plots (similar to map data)
rather than standard 1D chromatograms. The way I explain this to my students is
to imagine you are standing at ground level looking at a mountain range. You can
only see the peaks closest to you. Small peaks at the back may be hidden by large
ones at the front and even if the larger mountains are in the distance it may be hard
to fully distinguish them from smaller peaks in front. Such peaks may just look like
one mountain to the naked eye, just as two coeluting peaks can look like one larger
one in a standard HPLC trace.
Now imagine you are floating above the mountains looking down. Now you have
a contour map type view of a much larger area. You can see many more peaks going
in all directions, but you may be unable to tell the relative heights of each peak, and
smaller peaks may still be hidden by larger ones (foothills lost in the shadows of the
4.2 Displaying the Data 41
Fig. 4.1 Simplified plots showing chromatograms as single peaks (upper panel) and as a contour
plot (lower panel)
mountains so to speak). There may also be so many peaks visible that it is impossible
to count them all. A very basic illustration of this is given in Fig. 4.1.
Modern commercial 2DLC deconvolution software tends to take a series of slices
through the 3D landscape at different heights. The user can zoom in and out and also
generate a list of peaks that simply can’t be seen by eye using a single 3D map from
one perspective. There are also multiple alignment algorithms for the correction of
minor retention time shifts between multiple runs. But how do we get the map in the
first place?
The data obtained from an offline 2DLC separation consist of several single 1D
chromatographic files (one datafile per cut), which can be presented as an overlay
plot. With advanced computer software it is possible to convert the raw datafiles into
a matrix consisting of the first dimension, second dimension and signal intensity
values. This matrix then can then be converted to a visual form which usually takes
the form of a coloured contour plot with spots corresponding to the peaks of each
separated component. A colour gradient is then used to show the intensity of the
peaks as shown in Fig. 4.2.
42 4 Data Analysis
Fig. 4.2 Comprehensive, but un-optimised, 2D LC analysis of black tea. The same data are shown
as a contour plot (panel A) and a three-dimensional chromatogram (panel B)
The data can also be displayed as a 3D plot if desired (as seen in Fig. 4.2) however
this does not always make it easier to see the data. This means of course that any soft-
ware must be able to recognise which 2D peaks correspond to the same compounds.
This is especially relevant when one is interested in quantitative analysis. Under-
standing how data is processed and analysed is important because if you don’t under-
stand how the data was generated and displayed it is much harder to be certain of the
inferences you can draw from it.
4.3 Algorithms and Processing Methods for 2DLC Data 43
4.3 Algorithms and Processing Methods for 2DLC Data
The identification and counting of peaks in a 2DLC chromatogram are very impor-
tant, especially for large scale high throughput analysis. In such cases advanced
software and algorithms are needed. Chromatographic data are often pre-processed
for a number of reasons. These can be generalised as follows (Bos et al. 2020)
(i) denoising and smoothing
(ii) baseline (drift) correction
(iii) retention time alignment
(iv) peak deconvolution and resolution enhancement
(v) data compression
The Savitsky-Golay filter (Savitzky and Golay 1964) has become a universal
method to remove noise from scientific measurements. This algorithm not only
smooths noisy data but also provides the first to third derivatives of recorded chro-
matograms, which in turn are used to examine the shape of the chromatographic
curve, allowing overlapping and shouldering peaks to be distinguished and providing
retention and area information of LC peaks (Danielsson et al. 2002; Vivó-Truyols
et al. 2005). Many systems, such as the free to use XCMS software platform extracts
peak information in this way (Pierce and Mohler 2011). If you put your sample in
at the front and take your data out at the back, you have a limited idea of how the
data was generated nor if an alternative signal processing method might have been
better. I have often thought that analytical scientists could potentially learn a lot from
the signal processing advances made in electronic engineering in recent years that
enable internet and phone signals to be processed to such an extent that my voice
can travel almost instantaneously from one side of the world to the other and be as
recognisable, with no distortion as if I were standing right next to the listener.
Some researchers have tried alternative methods. The penalised least squares
smoothing algorithm which is based on the method first described by Whittaker
(1922) has been applied to 2DLC (Stevenson et al. 2013a, b) This method has the
advantage of not only reducing signal noise but also approximating the baseline
following an asymmetric least squares approach. Wavelet transforms have been used
to remove noise from HPLC and LC-MS data in the MetSign and OpenMS systems
(Tautenhahn et al. 2008; Wei et al. 2011) Interestingly, wavelet analysis has also been
applied to NMR spectroscopy data with some success (Rubtsov and Griffin 2007).
There are many other sophisticated chemometric analyses that can be used to
extract meaningful information from complex 2DLC data. Commonly used methods
are principal component analysis (PCA), partial least-square regression analysis
(PLS) (Tistaert et al. 2012) parallel factor analysis (PARAFAC) (Allen and Rutan
2011) or the Generalised Rank Annihilation Method (GRAM) (Ramos et al. 1987;
Sanchez et al. 1987). Most chemometric methods are used for the deconvolution of
overlapping peaks, reduction of dimensionality of an analyte of interest, and compar-
ison of samples, in addition to the improvement in data quality by removing artifacts
44 4 Data Analysis
or correcting the baseline and remove erroneous data (e.g. data generated from instru-
mentation spikes). Raw data can be analysed by dividing the separation area into bins
and calculating the area under the resultant curves (Pierce and Mohler 2011).
Techniques to extract peak information from traditional chromatographic spectra
can also be applied to 2DLC data. However, as several fractions per peak are trans-
ferred between dimensions a further processing step must be completed to assign
a single multidimensional retention time per eluted compound. Peters et al. (2007)
reported a protocol to combine features in neighbouring fractions by comparing the
peak start and end times measured from the second derivative of chromatographic
measurements; peaks were joined when the overlap surpassed a defined threshold.
Vivó-Truyols (2012) refined this process by joining peaks on the basis of their confor-
mation to Bayesian statistics. Similar processes have been applied by researchers
to assess similarities in retention characteristic between the two separation envi-
ronments (Stevenson et al. 2010) and find the cumulative area of 2DLC peaks by
fitting Gaussian, or exponentially modified, Gaussian models to each successive
chromatogram (Stevenson and Guiochon 2013).
In the methods discussed above each fraction is analysed as a discrete chro-
matogram and the data then looked at as a whole. Alternatively, 2DLC data can be
examined as a single three-dimensional dataset (where the dimensions are longitude,
latitude and altitude-going back to our mountain range metaphor) with the watershed
algorithm (Beucher and Lantuejoul 1979). This has been adapted by Reichenbach
and co-worker to 2DLC chromatograms and is available as the GC Image software
(Reichenbach 2009) which is relatively easy to use but also expensive. Essentially,
the watershed algorithm is used to find geographical pits. When applied to 2DLC,
inverted peaks are represented as basins that are subsequently filled with a hypo-
thetical fluid. An algorithm can then be applied that filters all catchment basins
that are below a given threshold thus creating a two-dimensional retention map.
However, it has been reported that the watershed method is limited in that it cannot
resolve the most complex datasets because it requires a crest between basins. This
means that it has limited ability to distinguish between severely overlapping and
shouldering peaks (Ramos 2009) and has been found to fail approximately 15%
of the time in GC × GC experiments (Vivó-Truyols and Janssen 2010). As noted
by Bos et al. (2020) the water-shed algorithm is often outperformed by the other
techniques, but it may find new application in the field of polymer analysis. Polymer
separations typically do not yield individually separated components (i.e. peaks),
but envelopes or distributions (sometimes called “smears”), which are difficult to
treat with curve-fitting or derivative-based methods.
Whatever software is used one must also be careful of the phenomenon known
as wrap-around. This occurs when the 2D retention time of a peak exceeds the
modulation period and so is dragged into the next modulation cycle. It is commonly
seen when a particular analyte possesses a very high affinity for the stationary phase
and hence moves slowly through the column. Wraparound causes the formation of
broader peaks, the width of which can mask other peaks from further modulations.
The distorted and larger peaks that result from wraparound are usually picked up
automatically by current software but are better prevented than cured.
4.3 Algorithms and Processing Methods for 2DLC Data 45
When peak information is in hand, chemometric processes can also applied to

2DLC measurements, usually to find changes in datasets. Principal component anal-
ysis (PCA) is a broad unsupervised approach to data analysis where samples are
grouped and aligned along axes representing the greatest differences in separation
behaviour (Eriksson et al. 1999) As such, it is a valuable tool to rapidly identify
similarities and differences between unknown datasets, or to confirm hypotheses
(Cook and Rutan 2014). Often studies are trying to find differences between two
distinct classes of sample, e.g. sick and healthy. In such cases, a supervised approach
can be taken such as partial least squares-discriminant analysis (PLS-DA) (Wold
and Andersson 1973). This has the benefit of training the algorithm with data from
known classes, which is then used to create models from linear combinations of
complex chromatographic data that differ from the provided quantitative informa-
tion; these models can then be used to classify new unknown data/chromatograms
but care must be taken not to overfit the data. Work on improved peak clustering
algorithm for comprehensive two-dimensional liquid chromatography data analysis
continues (Bos et al. 2020; Xu et al. 2019) and there is ample research space in this
area for the interested data analyst or chemometrician.
4.4 In Silico Method Optimisation
The use of statistical models to predict retention factors is well developed in chro-
matography, for example, the Kovats retention index used to convert retention times
into system-independent constants (Zellner et al. 2008). More recently in silico
screening has also been used to optimise the separation set up of complex 2D
separations such as the Ion Chromatography (IC) × Capillary Electrophoresis (CE)
separation of low molecular-mass organic acids (Ranjbar et al. 2017) and a blind
optimisation protocol for column selection from natural product extraction. In the
latter case a data processing pipeline, created in the open source ‘OpenMS’ software,
was developed to map the components within the mixture of equal mass across a
library of HPLC columns to find the best combination before the experiments were
run (Burns et al. 2016). This approach allowed for a significantly quicker selection
of two orthogonal columns.
A recent development in this area is the use of software, such as the commercial
program Drylab, to predict not only compound retention times but also the effects
that varying multiple parameters such as pH, temperature, and buffer concentration
will have on the retention time. This software allows the choice of columns and
solvents etc to be optimised in silico prior to running experiments (Andrighetto et al.
2014). Optimising experimental design in advance of running a physical experiment
has clear financial, temporal, and environmental benefits not least of which is the
avoidance of any potential need to waste precious samples.
Commercial software such as Drylab does come with a cost. Recently, however,
the C++ based MUSCLE (Multi-platform Unbiased Optimization of Spectrometry
via Closed-Loop Experimentation) software was created, which also allows the
46 4 Data Analysis
Fig. 4.3 Optimised model of caffeine interacting with an NVP monomer of Oasis HLB sorbent
in silico optimisation of LC separations (Bradbury et al. 2015). Although not as

advanced as Drylab, and reliant on the Windows operating system, MUSCLE has
the advantage of being free and open source. Both DryLab and MUSCLE can be
used to predict metabolite retention times given a certain set of operating conditions.
Theoretically, this means they could also be used to work backward and predict the
likely metabolite/chemical structure of an unknown peak from a chromatogram if
given the starting conditions—though this would take a lot of experimental work to
prove. Such automated software could also help with the active-modulation inter-
faces discussed in Chap. 3 where they would greatly reduce sensitivity effects and
solvent incompatibility.
Other research in this area is in computer-based modelling of sorbent/sorbate
interactions in chromatographic systems. An example of this is shown in Fig. 4.3.
Combining computational and experimental approaches was recently used to select
chromophores to enable the detection of fatty acids via HPLC, further demonstrating
the potential uses of this form of method development (Pandohee et al. 2019).
The new knowledge of such interactions could potentially advance our under-
stating of the underlying chemical theory and help further optimise 2DLC
separations.
4.5 Conclusions
Data analysis is a critical step in obtaining useful information from complex samples
using high throughput analysis and/or the increasingly advanced analytical tools such
as 2DLC. Many data processing methods in 2DLC are based on 1DLC methods or
4.5 Conclusions 47
techniques brought from 2DGC (2DLC’s bigger brother). In the future, methods,
such as peak alignment, for use in 2DLC are likely to be those designed specifically
for this technique (e.g. be designed to operate in two dimensions not just one). Data
processing methods are often difficult to compare however, because the results greatly
depend on the quality of the original data and the aim of the analyst. For example,
is the aim to separate as many compounds as possible or just to separate relatively
few (or even only one) peaks that are very hard to isolate? While useful results can
be obtained using today’s increasingly powerful commercial software the better the
user understands their sample, the aim of the experiment, how their data is pre- and
post-processed and presented the better the outcome will generally be.
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Chapter 5
Hyphenation
Abstract The identification of unknown compounds is of fundamental importance

for a range of applications in chromatography including, but not limited to, environ-
mental pollution, food/natural product analysis, metabolomics, sports drug testing
and petrochemicals, and biofuel analysis. Critical to the success of each such appli-
cation is the ability to separate the compounds of interest both from each other and
the sample matrix-which may be present at concentrations many orders of magnitude
higher than the target compounds. While two-dimensional chromatography increases
the available separation space there can still be problems identifying all the peaks in
a sample especially due to dilution of low concentration components in the second
dimension. Selectivity and sensitivity are key to solving such problems. Both may be
increased by the use of multiple dimensions of separation. In this chapter, some of the
methods of extending the power of 2DLC by hyphenating it with other dimensions
of separation are presented and discussed.
Keywords Collision cross section · Ion chromatography · Electrophoresis ·

Eluent · Ion mobility · Mass spectrometry
5.1 Background
If, as stated in Chap. 2 of this book, the best separations can be achieved using the
most varied columns as possible then could better separations be achieved using two,
totally different techniques? The short answer is yes but the long answer needs to
consider how these two systems can be connected. It could be argued that a truly
multidimensional system utilises two dimensions that have different mechanisms
of response, for example where a chemical separation dimension is coupled with a
spectroscopic dimension.
For any multidimensional separation system, the following conditions can
generally be thought of as applicable:
(i) The components of a mixture are subject to two or more separation steps, in
which their displacements depend on different factors.
https://doi.org/10.1007/978-981-15-6190-0_5
50 5 Hyphenation
(ii) When two or more components are separated in any single step, they always
stay separated until the total separation operation is finished (Kouremenos et al.
2016).
To respond to these challenges many analysts utilise advanced mass spectrom-
etry (MS) and data processing software to deconvolute complex overlapping chro-
matograms. This approach is not always optimum. No software is perfect and mass
spectrometry has also been accused of telling you the answer you want, not the
answer you need. Sometimes compound specific detectors such as electron capture
(for halogens) or the nitrogen–phosphorus detector (NPD) may give more definitive
results.
It may not be too unfair to say that some hard-core (and perhaps even some not
so hard core) chromatographers regard mass spectrometry as just another detector
while some mass spectrometrists tend to think of chromatography as just a fancy
method of sample preparation. In truth, both sides have a point, and both also need
to see the wider picture and work together, not separately. Chromatography is more
than sample preparation and mass spectrometry (not spectroscopy!) is a valid form
of separation science that separates on mass/charge ratio rather than retention time.
The two techniques provide complementary information. LC-MS is the current gold
standard in analytical chemistry and so LC × LC-MS is likely to be even more useful
in many cases.
Hyphenation of LC to MS somewhat relaxes the demands placed on chromato-
graphic separation of species because of the inherent selectivity of MS detection,
especially in the form of tandem or high-resolution (HR) MS. However, MS also
suffers from some limitations, such as distinguishing between isomeric species and
its susceptibility to matrix effects. Chemically sensitive detectors and extra dimen-
sions of separation are other options but there is no perfect method. The simplest way
to identify an unknown compound is still to first separate it from other compounds
in the sample. This is, of course, the whole point of two-dimensional liquid chro-
matography, but can we do more, and if so, what are the best dimensions to add to a
2DLC system? One way to answer this question might be to try and conceptualise
where the overlap between the various forms of separation techniques might be.
Figure 5.1 illustrates a conceptual “separation space” that encompasses the
majority of relevant chromatographic techniques that could be hyphenated together
and illustrates the extent of the distinction and overlap among them. It is based on
the concept originally proposed in Talebi et al. (2016).
5.2 Mass Spectrometry
I have noted elsewhere in the book (but it is important so bears repeating) that there is
no one best method of analysis since the best method to use depends on the aim of the
analyst. If your aim is to identify as many things as possible as quickly as possible the
one “obvious” extra dimension to add to a 2DLC system is mass spectrometry, which
5.2 Mass Spectrometry 51
Fig. 5.1 An illustration of a conceptual separation space adapted from Talebi et al. (2016)
comes in almost as many varieties as chromatography. Which is best? Well, resolving

power is crucial in 2DLC and this is where MS with fast scan speed (such as time
of flight MS) can be very helpful. One form of spectrometry that shows particular
use in this area is ion mobility-spectrometry mass spectrometry (IMS) because it
adds together separation by mass and by drift time. Indeed linking ion mobility with
two dimensional chromatography can give four dimensions of separation and such
systems are currently starting to appear in the literature linked to both GC × GC
(Lipok et al. 2018) and LC × LC systems (Stephan et al. 2016a).
For those unfamiliar with the technique, IMS separates ions drifting through a
tube filled with buffer gas in a weak electric field according to their shape-to-charge
ratio (rather than mass to charge in standard MS) (Kanu et al. 2008). This gives
IMS the ability to measure collision cross sections (CCS) of the ions in question.
The CCS is a specific value for an ion and can act as an additional parameter to
exact mass, increasing certainty in the identification of compounds. A very handy
application of CCS and IMS is that it provides the analyst with the ability to separate
isobaric species (ions with identical masses but different atomic compositions) when
they differ in their size and/or shape. Even the best high-resolution MS would deliver
only one peak for two isobaric ions because they have the same mass-to-charge (m/z)
ratio. The separated ions are then introduced into a mass analyser in a second step
where their mass to charge ratios can be determined on a microsecond timescale.
Drift time can then be added to retention time in the analysis. Comprehensive two-
dimensional liquid chromatography (LC × LC) and ion mobility spectrometry–mass
52 5 Hyphenation
spectrometry (IMS–MS) are increasingly being used to address challenges associated

with the analysis of highly complex samples (Causon and Hann 2015). The advantage
of ion mobility for characterizing unknown compounds by their CCS and accurate
mass in a non-target approach is not insignificant.
Venter et al. (2018) evaluated the potential of a comprehensive three-dimensional
(HILIC) × reversed phase LC (RP-LC) × IMS–high-resolution MS to analyse a range
of phenolic compounds, including hydrolysable and condensed tannins, flavonoids,
and phenolic acids in several natural products. A protocol for the extraction and
visualization of the four-dimensional data obtained using this approach was devel-
oped as part of the process. The benefits associated with the incorporation of IMS
include improved MS sensitivity and mass-spectral data quality. IMS also provided
separation of trimeric procyanidin isomeric species that could not be differentiated
by HILIC × RP-LC or HR-MS. The performance of the LC × LC × IMS system was
characterised in terms of practical peak capacity and separation power, using estab-
lished theory and taking undersampling and orthogonality into account. Overall an
average increase in separation performance by a factor of 13 was found when IMS was
incorporated into the HILIC × RP-LC–MS workflow. The authors did note however
that IMS separation performance and the extent of second dimension undersampling
depended on the upper mass scan limit, which might present a limitation for the
analysis of larger molecular ions such as proteins or polymers.
Stephan et al. (2016b) used LC × LC, in combination with ion mobility-high-
resolution mass spectrometry to separate components of various complex samples
in four dimensions. Their system worked as a continuous multi heart-cutting LC
system, using a long modulation time of 4 min. This allowed the complete transfer
of most of the first-dimension peaks to the second-dimension without fractionation
meaning that each compound showed up as only one peak in the second dimension.
This is useful as it simplifies the data handling even when ion mobility spectrometry
as a third and mass spectrometry as a fourth dimension were introduced. The authors
tested the system on the analysis of a plant extract and showed the separation power
of this four-dimensional separation method had a potential total peak capacity of
>8700.
Natural products such as those used in the two studies detailed above are often
used to test the effectiveness of separation techniques due to their complex sample
matrix and the fact that such samples usually contain not only a large number of
very similar individual compounds but also a large number of compound classes
which can vary by orders of magnitude in size, concentration and polarity (McCance
et al. 2018). Natural products are complex samples to be sure, but they are not
the only ones. Comprehensive analysis has also become an important tool in the
field of environmental science and toxicology since a huge variety of pollutants
from different sources are can be found in the environment, ranging from pesticides
and pharmaceuticals (Pérez and Barceló 2007) to perfluorinated compounds such
as per-and poly-fluoroalkyl substances (PFAS) as well as disinfection by-products
(Alexandrou et al. 2019). Liquid chromatography coupled to high-resolution mass
spectrometry is currently the gold standard for the identification of such complex
5.2 Mass Spectrometry 53
mixtures of compounds. Non-targeted analysis of a wastewater sample with a four-

dimensional separation platform with HPLC coupled to an ion mobility-quadrupole-
time of flight mass spectrometer (IM-QToF-MS) revealed 53 different compounds,
identified by exact mass and CCS, in the examined wastewater sample. Fifty-three
compounds may not sound like a huge number but critically this method was able
to distinguish between isobaric structures such as cyclophosphamide and ifosfamide
which would have been impossible to do with standard MS.
5.3 Multiple Chromatographic Methods
Many analysts will be familiar with normal phase (NP) and reverse phase (RP)
HPLC. However, there are multiple forms of chromatography and nearly all
have been combined at some point to explore approaches to LC × LC. These
including reversed-phase liquid chromatography (RPLC) coupled to size-exclusion
chromatography (SEC)(Li et al. 2015), RPLC to RPLC (Donato et al. 2011), ion
chromatography (IC) to SEC (Xu et al. 2018), IC to RPLC (Brudin et al. 2010)
ion-exchange chromatography with a strong cation-exchange column (SCX) to
RPLC (Kajdan et al. 2008), normal-phase liquid chromatography (NPLC) to RPLC
(Wei et al. 2009), supercritical fluid chromatography to RPLC (Yang et al. 2020),
hydrophilic interaction chromatography to RPLC (Cao et al. 2018), and even IC to
IC and CE (Fa et al. 2018). Most of these tend to be rather specialised applications
and the most common combination of 2DLC is RPLC × RPLC (Pirok et al.
2019)-although the combination of HILIC and RP provides powerful separation
capability for the analysis of polar compounds in samples with complex matrices.
Despite high orthogonality, on-line combinations of organic normal-phase and
reversed-phase chromatography are less frequent, due to the limited mobile phase
compatibility. Hence, NP systems are usually combined with RP systems off-line.
The compatibility problem is less prohibitive, though it is still important, with
direct coupling of reversed-phase and hydrophilic interaction liquid chromatog-
raphy, HILIC, (aqueous-organic normal phase) systems (RP × HILIC, HILIC ×
RP). Because of good compatibility, the combination of the same principles, but
with different stationary and mobile phases, are popular in RP × RP based methods.
Although it is more difficult to select fully two-dimensional systems with a high
degree of orthogonality when the same format is used in both dimensions it is possible
to increase orthogonality by utilising a different pH in each column.
Generally, there are no significant problems with mobile phase compatibility in RP
× RP, RP × IEC, SEC × RP or SEC × NP two-dimensional LC separation systems.
In contrast it is usually not easy to couple organic normal-phase and reversed-phase
modes on-line, because of mobile phase immiscibility. NP systems should generally
not be used in the second dimension as water transferred in aqueous-organic mobile
phases from the first, RP, dimension may deactivate the polar adsorbent used for
NP separations in a non-aqueous mobile phase and destroy the separation. Further,
54 5 Hyphenation
large viscosity differences between the mobile phases used in the two dimensions
may distort peak shape due to flow instability (viscous fingering effect).
Occasionally, the system incompatibility problems can be less important when
an organic NP system is used in the first dimension and the RP system in the
second dimension. Anyway, RP-NP systems are usually connected off-line. For
on-line connection, an interface was recently introduced, in which volatile organic
mobile phase is evaporated from the first-dimension NP fractions, before introduc-
tion into the second, RP dimension, often at a cost of incomplete recovery of sample
compounds, especially those with low boiling points. The system compatibility prob-
lems are avoided when non-aqueous reversed-phase chromatography (NARP) is
combined with normal-phase chromatography, in a 2D NARP × NP LC on-line
setup but this solution can usually be used only for low-polarity samples, such as
lipids.
Polar compounds can be separated in coupled reversed phase × HILIC systems,
which often show complementary selectivity. The mobile phases used in HILIC
contain low concentrations of water in an organic solvent such as acetonitrile and are
well miscible with aqueous-organic mobile phases used in both RP × HPLC and RP
× HILIC combinations still present significant elution strength mismatch problems.
Highly organic HILIC mobile phases are strong eluents in the reversed-phase
systems and if transferred as the fraction solvent into the RP second dimension, they
may significantly deteriorate the band shape and resolution. This also applies for
RP × HILIC setups, as the RP mobile phases usually contain water concentrations
high enough to interfere with the HILIC separation in the second dimension.
None of the combinations listed above are likely to offer true orthogonality in
the mathematical sense (i.e. being statistically independent) although some of them
come very close. They also come with specific problems. Ion chromatography usually
involves KOH for example, and this is not compatible with RP or MS systems.
Linking IC to MS is made possible however by neutralising the first-dimension
effluent, containing KOH, before transferring it to the second-dimension reversed-
phase column (Brudin et al. 2010). Similarly linking RPLC to NPLC requires modi-
fication of mobile phases to ensure they are compatible between columns. This can
be done of course and the major driver to apply 2DLC methods to maximize peak
capacity to tackle complex samples means that even more novel combinations of
chromatography columns are likely in future.
5.4 Combining Chromatography and Electrophoresis
Multidimensional electrophoretic and chromatographic separations can be powerful

complementary techniques for the analysis of lower molecular mass molecules, in
addition to proteins and peptides. The first comprehensively coupled multidimen-
sional separation approach combining liquid chromatography and capillary elec-
trophoresis (LC × CE) was described by Bushey and Jorgenson (1990). This work
offered remarkably enhanced resolving power compared to its single dimensional
5.4 Combining Chromatography and Electrophoresis 55
building blocks. Since this time there have been a surprisingly large number of 2D
separations with LC and CE (perhaps this is only surprising only you don’t work
with CE) and these were recently detailed in an excellent and comprehensive review
by Ranjbar et al. (2017).
In the environmental space, a recent 2D separation was accomplished using
a comprehensively coupled online two-dimensional ion chromatography-capillary
electrophoresis (IC × CE) system. The system was successfully used to resolve a suite
of haloacetic acids, dalapon, and common inorganic anions in water. The system was
custom built and employed a sequential injection-capillary electrophoresis instru-
ment and a non-focusing modulation interface, comprising a tee-piece and a six-port
two-position injection valve, to allow comprehensive sampling of the IC effluent.
High electric field strength (+2 kV/cm) enabled rapid second-dimension separations
in which each peak eluted from the first-dimension separation column was analysed
at least three times in the second dimension. Two-dimensional peak capacity was
498 with a peak production rate of 9 peaks/min.
In the biochemical area there have been some novel approaches to linking IC to
CE coupled with mass spectrometry (IC × CE-MS) to study ionic metabolites such
as the organic acids and phosphorylated species that comprise glucose metabolism
and the tricarboxylic acid (TCA) cycle (Burgess et al. 2011; Ramautar et al. 2017).
The disadvantage of these systems is they are complex to run and require on-line
desalting to turn high salt eluents from the IC step into MS compatible water, but
this is not impossible to do with modern chromatographic technology (Beutner et al.
2018). Stationary phase-assisted modulation (as discussed in Chap. 3) can help in
this regard as it allows salts or organic solvents from a first-dimension ion-exchange
separation to be removed before aqueous reverse-phase LC separation using MS.
A major drawback to widespread adoption of this technology is that libraries of
standard compounds are currently lacking for IC and CE-MS systems.
Notwithstanding the seemingly ideal mechanistic considerations, there are
numerous practical constraints impeding LC × CE. Effective transfer of LC effluent
to CE is the main challenge to address when coupling the two techniques but other
issues include the fact that the typical peak volume in LC is substantially larger
than the injection volume in CE and the hydrodynamic flow in LC affects the elec-
trophoretic separation. As a result of complicated interfacing, LC × CE has not
experienced the wide employment or development of GC × GC or LC × LC. But
what about linking LC to GC?
5.5 LC × GC
The linking of an LC to a GC system was first published by Majors in 1980, who

used it to determine anthrazine in a sorghum sample via regular vaporisation and
reinjection of a small part of the HPLC fraction onto a GC (Majors 1980). LC-GC
systems have been studied since the 1990s primarily by Grob and Biedermann in
Switzerland and the Mondello group in Italy (Biedermann and Grob 2012; Grob et al.
56 5 Hyphenation
Fig. 5.2 A view of GC column (left) and the silica particles that are tightly packed in LC columns
(right) under an electron microscope to illustrate some of the differences between the two techniques.
Images created at the RMIT University Microscopy and Microanalysis Facility (RMMF)
1991; Mondello et al. 1999; Zoccali et al. 2015). They have primarily been used for
the analysis of food contact materials and these studies have been reviewed recently
(Biedermann and Grob 2012). Such systems can be used for more than just food
testing. LC-GC × GC is also attractive for trace analysis of pesticides for example
due to the excellent detection limits afforded by large volume injection and the lack
of sample pre-treatment, which may not extract all pesticides in a sample (Godula
et al. 2001).
It should be noted, however, that the coupling of LC to GC is not a trivial issue,
as both the LC and GC operate with mobile phases that are in two different phys-
ical states, at different pressures and which contain different additives. Some of the
differences between the two are shown in Fig. 5.2.
Normal-phase liquid chromatography is more easily coupled with GC than
reversed-phase because the eluent is usually a non-polar volatile solvent and thus
NP based LC tends to be the favoured method.
There are two possibilities to transfer the LC eluent to a GC. One must either
regulate the LC flow according to the requirements of the two-dimensional GC
unit, which necessitates using µ-LC, which in turn may limit sample detection,
or introduce very large volumes into the GC. The latter is usually preferable as it
allows for lower detection limits. A good guide to how to set up such a system is
given in Kouremenos et al. (2016).
There are also (very complicated) commercial systems such as the Shimadzu 5D
Ultra-e LC-GC × GC-MS/MS system which offers five dimensions of separation.
This is perhaps a little over the top and not needed outside very specialised applica-
tions. In general LC × GC (whether linked to MS system or not) is still considered
a complex and specialised technique, so few laboratories use it continually and the
applications to date have been limited. The future of LC-GC × GC will rely on (i)
5.5 LC × GC 57
available and easily implemented user-friendly technology, (ii) more reported appli-
cations, and perhaps most crucially (iii) the development of proper data handling
techniques.
5.6 Conclusions
Recent developments in modulation technology and automated computational

method optimisation have unlocked new possibilities in hyphenation of different
forms of LC. RPLC × RPLC is currently the most used form of LCLC with HILIC
gaining popularity, boosted somewhat by the fact that new modulation techniques
can overcome solvent incompatibilities and sample dilution in the second dimension.
The combinations of new column chemistries that could be used are many and varied
and the use of size exclusion and chiral columns in the first dimension offers up many
further opportunities for novel applications. Novel hyphenations such as IC × CE
and LC-GC are still rather specialist tools but show promise. Ultimately the use of
novel hyphenations to push the boundaries of separation science can only be for the
better.
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Godula M, Hajšlová J, Maštouska K, Křivánková J (2001) Optimization and application of the PTV
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Chapter 6
Applications of 2DLC
Abstract It has been shown that two-dimensional liquid chromatography offers a

way to greatly increase the number of compounds that can be separated, detected
and quantified in a single analytical run. The potential of 2DLC is high but to gain
more widespread acceptance by academia and especially industry it is necessary to
show that multidimensional LC can be applied to real-world analytical problems
and produce useful results. This chapter therefore gives an overview of some of the
many (and growing) applications that admirably demonstrate the utility of 2DLC,
including pharmaceutical analysis (for both small molecules and biopharmaceuti-
cals), natural product chemistry, polymer analysis, forensic sciences and the ‘omic
sciences (metabolomics, lipidomics and proteomics).
Keyword Forensics · Natural products · Lipids · Pharmaceuticals · Polymers ·

Proteins
6.1 Background
Now that we have understood the background and theoretical underpinnings of 2DLC
let’s cover some of its potential applications. Note that we won’t go into all potential
applications here as that would very easily be a book in its own right.
All fields in which both high peak capacity and high selectivity are needed
could benefit from multidimensional LC. These include, but are not limited to, the
following.
• Pharmaceuticals (including biopharmaceuticals)
• Natural products and herbal medicines
• Omic sciences (metabolomics, lipidomics, proteomics)
• Polymers (including surfactants)
• Forensic science and toxicology
• Environmental science and ecotoxicology
https://doi.org/10.1007/978-981-15-6190-0_6
62 6 Applications of 2DLC
2DLC could also be used for the analysis of fuels and related petrochemicals
although these types of samples are already well served by 2DGC (Frysinger et al.
2002; Giri et al. 2017; Pandohee et al. 2020).
It would be impossible to list all the applications here and in some sense it would
be redundant to do so since there is a comprehensive list of applications that can
be accessed for free online https://www.morepeaks.org/pirok/2dlc-applications.php
and http://multidlc.org/literature/2DLC-Applications. We will, however, have a look
at some examples in the literature to gain some idea of what is possible with the
technique.
6.2 Pharmaceuticals
Many pharmaceutical products are highly complex mixtures that may contain
multiple Active Pharmaceutical Ingredients (APIs). A single drug product may
contain both the active ingredient and impurities. There may be APIs with multiple
chiral centers, products that contain both small and large molecules (e.g., protein-
drug formulations with surfactant excipients) and drug products with impurities that
need to be identified and removed. The use of an achiral × chiral column setup would
be ideally suited for the analysis of impurities in chiral pharmaceutical substances for
example. It is likely that 2DLC will play an important role in this area in future. For
example, heart-cutting with a 2DLC-MS platform enables characterization of mono-
clonal antibodies and antibody-drug-conjugates during all stages of the product life
cycle (Largy et al. 2016; Ouyang et al. 2015; Williams et al. 2017).
In recent years 2DLC has been used in pharmaceutical analysis to address chal-
lenges such as resolving peak co-elution, impurity quantification, peak purity assess-
ment, stability/degradation testing, and chiral separations (Zhang et al. 2013). Kiffe
et al. (2007) for example, compared both one and two dimensional liquid chro-
matography (in the offline mode) in conjunction with MS to analyse urine and faeces
from rats and mice for the structural elucidation of metabolites from drug candi-
date compounds during absorption distribution, metabolism and excretion (AMDE)
studies. This work may not sound glamourous, but it is essential for pharmaceutical
development. The research also made use of heart cutting to transfer fractions of
interest to a second dimension and is worth including here since the compounds of
interest had to be separated out from complex biological matrices in the same way
as biological metabolites are.
The radiolabelled version of the compound being assessed was created and fed to
the test animals. Urine and faeces were then collected, and the metabolites extracted.
The samples were then run on standard one-dimensional liquid chromatography.
Effluent from the first dimension was then collected in 96 well plates and poten-
tial metabolites were located by testing each fraction for radioactivity. A positive
result indicated that the fraction held either the radiolabelled parent compound or
a radiolabelled metabolite derived from it. The fraction with radioactive metabolite
fractions was then injected onto a second LC system with hyphenated MS detection
6.2 Pharmaceuticals 63
for the identification and subsequent structural determination. The study showed
that applying the 2DLC-MS approach enabled characterisations of major, minor,
and trace metabolites and was a useful method to separate out radiolabelled mate-
rials from all the other compounds present in a particular biological sample. Such
a method could be used to track many different compounds though many complex
biochemical pathways.
6.3 Natural Product Chemistry
Natural products chemistry is an old technique that has undergone somewhat of a

resurgence in recent years. Today it is a major resource for the study of naturally
occurring biologically active substances. The traditional way of studying natural
products is pre-treatment and fractionation of a complex matrix followed by sepa-
ration and isolation of the individual components using repeat liquid/column chro-
matography, in an off-line mode. The off-line approach is very easy but presents
several disadvantages: it is time-consuming, operationally intensive, and difficult to
automate and in some cases difficult to reproduce. There may be hundreds if not
thousands of possible compounds isolated from natural products which will vary in
size, polarity and stability. The natural product chemist may be interested in one or
two such compounds or a class of very closely related compounds.
Qiu et al. (2014) created an on-line comprehensive medium pressure liquid chro-
matography (MPLC) × preparative LC system connected using a solid phase trapping
column. The system allowed for the automated multi-step preparative separation of
25 compound with enough of each that their structures could be identified by MS, and
both proton and carbon NMR. The 2DLC approach, therefore, offered great advan-
tages in analytical efficiency and sample treatment capacity compared with conven-
tional methods. Since this study was published there have been numerous reports
of similar methods of sample enrichment and comprehensive characterization (Liu
et al. 2019; Zhao et al. 2019).
In a similar vein to natural product chemistry, the enhanced peak capacity and reso-
lution of 2DLC has led it to be used in the profiling of Chinese medicines including
the development and introduction of novel stationary phases for such research (Zhou
et al. 2020). Indeed, one of the most common untargeted LC × LC applications is the
profiling of natural products in plant extracts, particularly those used in traditional
Chinese medicines (Brandão et al. 2019). In both areas the huge number of possible
(but in many cases unknown) compounds that might be of interest/responsible for a
supposed therapeutic effect makes the extra separation space of 2DLC particularly
valuable.
6.4 Metabolomics
Metabolomics is the integrated analytical biochemistry and bioinformatics based

study of the biological metabolites. It involves profiling as many small molecules as
possible in a biological system through large-scale, non-targeted or targeted, high
throughput determination of metabolites in a biological system. The challenge of
metabolomics is to comprehensively cover the analysis of as many compounds as
possible. This is hindered by the wide chemical diversity (size, weight, polarity,
stability) of metabolites in a sample, high availability of abundance, and establish-
ment of the different approaches for extraction, separation, detection, and quanti-
tation, and identification of novel compounds. Recent work on the application of
2DLC to metabolomics has shown that it can analyse both aqueous and organic
phase metabolites in one sample run. A good review of 2DLC in metabolomics can
be found in Pandohee et al. (2015b) but a few examples are discussed below.
Fairchild et al. (2010) used off-line 2DLC with tandem mass spectrometry detec-
tion (2DLC/MS-MS) to assess aqueous metabolites extracted from Escherichia coli
and Saccharomyces cerevisae cultures using an SCX × HILIC column set up. They
developed a single set of chromatographic conditions for both positive and negative
ionization modes and were able to detect a total of 141 metabolite species (92 for
E. Coli and 95 for S. cerevisae with 46 in common). The method gave an overall
peak capacity of approximately 2500. The work demonstrated that a single two-
dimensional separation method is sufficient and practical when a pair or more of
one-dimensional separations are used in metabolomics studies. In order to obtain
sample high throughput, the authors relied upon what they described as a ‘workhorse
approach’ in which the first dimension was short and only a small number of fractions
were collected. The second dimension and the detector were relied upon for much
of the separation power which in turn meant that the total peak capacity was lower
than it might have been since some chromatographic performance was sacrificed to
reduce the overall analysis time.
Pandohee et al. (2015a) also used the offline 2DLC approach with a diode
array detector to study how the metabolic profiles of Agaricus bisporus mushrooms
changed upon UV exposure. The authors tested several metabolite extraction methods
and then used selectivity studies to find the most orthogonal columns for the analysis,
settling on a Cyano and a C18 for the first and second dimensions, respectively. The
data were processed using custom written code in Mathematica. The method allowed
the detection of 158 peaks from several different compound classes including sugars,
amino, organic and fatty acids and phenolic compounds in a single analytical run;
although only 51 of these could be identified. The study gives a good overview of the
2DLC method development process and demonstrates the increased peak capacity
and separation space of 2DLC and its potential in metabolomics since the method
was able to identify a wider array of compounds than 1DLC, nuclear magnetic reso-
nance spectroscopy or GC-MS. The run time, however, was several hours per sample
and thus the method had the potential to be improved via the use of online 2DLC.
6.4 Metabolomics 65
Fully automated online 2DLC was used by Klavins et al. (2014) in conjunc-
tion with tandem mass spectrometry (MS/MS) to detect and quantify sugar phos-
phates in cell extracts from the methylotrophic yeast Pichia pastoris. An anion
exchange column was used as the first dimension and porous graphitized carbon
as the second. This particular study used the heart cutting technique and careful
method development enabled chromatographic separation on the second dimen-
sion to be optimized for each transferred fraction, thus minimizing the sepa-
ration time and ensuring complete removal of the salt constituents of the first
column eluent. The method gave sub-µM limits of detection ranging between
0.03 and 0.19 µM for the investigated compounds. A total of 10 sugar phos-
phates (glucose-1-phosphate, glyceraldehyde 3-phosphate, glucose-6-phosphate,
mannose-6-phosphate, fructose-6-phosphate (F6P), ribose-5-phosphate, sedohep-
tulose 7-phosphate (S7P), 3-phosphoglyceric acid, 6-phosphogluconic acid and
fructose-1,6-bisphosphate) were identified and perhaps, more crucially, were quanti-
fied by this technique. At first glance a method identifying only ten compounds does
not sound like it is taking full advantage of the 2DLC technique, but quantification
of sugar phosphates is a very complex task. The ability to separate the phosphates
of interest from the hundreds of other compounds in a typical yeast extract is of
high importance, and this method combined the efficiency of strong anion exchange
(SAX) chromatography with the selectivity of MS/MS detection.
6.5 Proteomics
Proteomics is the analysis of all the proteins in a particular sample. Research in

this area involves the analysis of very complex samples, often comprising hundreds
of thousands of proteins or peptides which may be fragmented or modified (e.g.
phosphorylated or glycosylated) versions of the parent compound. Because the LC
separation and MS identification of intact proteins are difficult, the LC−MS anal-
ysis is typically performed on a peptide level, after protein digestion with suitable
proteolytic enzymes. This further increases the sample complexity and reinforces
the demands for efficient separation methods which makes 2DLC very useful.
There are huge numbers of papers on 2DLC proteomics. The most common 2D-
LC setup for proteomics is a combination of either SCX chromatography with RP
or RP × RP-LC but this may not suit everybody and a large challenge for proteomic
applications is still working out the most orthogonal column set to use. Fortunately,
the peptide separation orthogonality for 16 different 2D LC-MS systems has recently
been determined using a retention data set of ~ 30 000 tryptic peptides for each 2D
pairing (Yeung et al. 2020). This study noted that separation orthogonality generally
increases in the order RP < SCX < HILIC < SAX, with the exception of high pH
RP–low pH RP system, which showed the second-best orthogonality. A good review
of multi-dimensional liquid chromatography in proteomics can be found in Zhang
et al. (2010).
6.6 Lipidomics
Li et al. (2014) made use of online 2DLC, coupling normal and reversed-phase
columns to a quadrupole time-of-flight mass spectrometer (QToF-MS) and applying
the method to the comprehensive profiling of lipids in human plasma. This method
enabled the determination of 540 endogenous lipid species from 17 classes with the
limit of detections of 19 validation standards in the ng/mL range. The authors also
assessed the difference in lipid metabolism products from healthy individuals and
those suffering from atherosclerosis. Levels of galactosylceramides in atherosclerosis
patients were 1.5–2.8-fold higher than glucosylceramides in atherosclerosis patients
compared to controls. Although the sample sizes were small (there were only 12
subjects in total), the study demonstrates the potential of 2DLC to both lipidomic
(and atherosclerosis) research.
More recently a novel online two-dimensional supercritical fluid
chromatography/reversed-phase liquid chromatography–triple-quadrupole mass
spectrometry (2D SFC/RPLC-QQQ MS) method using a vacuum solvent evapora-
tion interface was developed and tested for use in lipid profiling of human plasma
(Yang et al. 2020). This approach allowed lipid classes to be separated by Super
Critical Fluid Chromatography (SFC) and then different lipid molecular species
within those classes were further separated by the second-dimension RPLC. The
method allowed the identification of 370 endogenous lipid species from ten lipid
classes, including diacylglycerol, triacylglycerol, ceramide, glucosylceramide,
galactosylceramide, lactosylceramide, sphingomyelin, acylcarnitine, phosphatidyl-
choline, and lysophosphatidylethanolamine, in human plasma to be separated within
38 min. The limit of detection was in the order of nanograms per millilitre. This is a
nice approach, but not every lab has SFC of course.
6.7 Environmental Science
One dimensional LC-MS (particularly QQQ mass spectrometry) is currently the

gold standard in environmental laboratories worldwide due to its sensitivity and the
fact that it is not limited to (semi) volatile compounds (or compounds that can be
made volatile by chemical modification) as GC-MS is. It may be surprising therefore
that 2DLC has not yet been widely used in environmental science. This is probably
explained by the fact that environmental samples can be very dirty. Wastewater
and river water samples can have high sediment and particulate loads and/or large
compounds such as fulvic and humic acids. Particulates can be filtered out of the
sample but if the compounds of interest are bound to these substances they will not
be seen in the final analysis. Large substances that give colour to the sample can
interfere with ionisation in the mass spec and removing them can also remove the
compounds of interest.
6.7 Environmental Science 67
There have been two studies using 2DLC with environmental samples and both
involved online RPLC × RPLC coupled to MS to assess the potential for semi-
targeted analysis of test wastewater samples. These studies allowed for the identifi-
cation of 23–65 compounds, including analgesics such as paracetamol and tramadol,
the herbicides diuron and monuron, benzotriazole (a corrosion inhibitor), and antide-
pressants such as venlafaxine and sertraline (Haun et al. 2013; Leonhardt et al. 2015).
This perhaps does not sound like a lot, but the ability to detect multiple compound
classes at ng/L concentrations quickly is a great starting point for this area. 2DLC
has also been applied to look at the molecular size continuum and associated light-
absorption properties of chemically distinct pools of urban organic air particles (Paula
et al. 2016) showing that it can contribute to the understanding of more than just the
aquatic compartment of the environment.
6.8 Forensic Toxicology
The focus of a forensic toxicology laboratory is generally to determine the pres-

ence or absence of drugs in biological samples such as urine, blood, oral fluid, or
hair, to see if an illicit drug or toxin played a role in a person’s death. The sensi-
tivity of the analytical method is critical because scientists need to detect chemical
compounds in very small amounts. A particularly tricky issue in this regard are novel
psychoactive substances (NPS). Specific NPSs are banned but illicit drug manufac-
turers often respond by altering the NPS chemical structures to make new (but similar)
compounds. A large range of analogues can be made in this way (Plummer et al.
2016). This not only circumvents legislation (unless an entire class of compounds
is banned rather an individual compound) but each time a new structure is intro-
duced, there is a possibility that it has not been previously recorded in mass spectral
databases. As analytical chemists, we know that you generally only see what you
look for. So if you use targeted analytical methods that rely on libraries of known
compounds to identify drugs in samples you may well miss new compounds that
may or may not coelute with known substances. The use of 2DLC to separate and
identify multiple compounds that may have very similar properties is of great poten-
tial benefit in forensic toxicology. High-resolution mass spectrometry is a good way
to screen a wide variety of analytes because of its high sensitivity and mass accuracy
but you still need to separate the isomeric and/or structurally similar compounds out
from each other. RP × RP systems have been used for the separation of co-eluting
and isomeric synthetic cannabinoids in blood and urine (Eckberg 2018) and for the
detection of cocaine and metabolites in bone (Mella et al. 2017), to the parts per
billion level.
6.9 Polymer Science
The characterization of complex polymers that feature multiple, independent distri-

butions is an obvious fit for LC × LC because of the large number of structurally
related compounds coupled with the difficulty of applying MS for characterizing
high-molecular-weight polymers. Indeed, polymer science is probably the second
most common application in 2DLC after proteomic analysis (although one could of
course argue that proteins are a type of polymer). 2DLC has been used to look at
many applications from complex mixtures to block co-polymers (Im et al. 2007).
Many 2DLC analyses of synthetic polymers have employed SEC for the second
dimension analysis due to the relatively short run time in addition to its wide use in
polymer analysis. By virtue of high temperature operation (leading to lower solvent
viscosity and high diffusivity of the polymer molecules), some authors have been
able to use normal length SEC column at a high flow rate with little loss in resolution
(Im et al. 2009). Reliable SEC separations of polymers with molecular weights
up to ca. 50 kDa can potentially by achieved in less than 1 min at pressures of
about 66 MPa (Uliyanchenko et al. 2011). However, this also means some quite
labour-intensive work, especially for some new polymer systems that require more
exotic separation conditions, such as high temperatures, toxic eluents and the use
of salts in, for example, SEC. All of this makes fractionation and especially sample
prep of the fractions even more complex and in many cases the traditional off-line
approach is still more efficient. Semi-preparative fractionation in the first dimension
and reinjection (after elimination of the first-dimension solvent and some further
sample prep) on the second dimension is usually helpful, especially as the resulting
fractions can still then be taken for further analysis via NMR or differential scanning
calorimetry (DSC).
6.10 Conclusions
Although not yet widespread the increased separation power of two-dimensional

chromatography is of great potential in a large variety of scientific areas, particularly
for compounds that are too sensitive for mass spectrometry but contained in matrices
too complex for standard LC analysis. The combinations of new column chemistries
that could be used are many and varied and the use of size exclusion and chiral
columns in the first dimension offers up many further opportunities for novel appli-
cations. Since 2DLC technology is now relatively easy to run and the high-pressure
capability of modern instrumentation allows high flow rates and correspondingly
high-speed analysis (and one no longer has to count individual peaks by hand) the
number of applications of the field is likely to continue to grow.
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Chapter 7
Conclusions and Future Developments
Abstract There has been a major shift in how multidimensional LC is perceived in

the last few years. Many more analysts are aware of 2DLC, but it is still not a routine
technique. More attention to robustness and method development strategies is still
required, but 2D-LC is no longer a technique that suffers from solvent-compatibility,
cumbersome method development, and reduced detection sensitivity. Instead, it is
a rapidly maturing technique with clear applications in academia and industry. But
how can we move it forward and what might the future hold? In this chapter we will
discuss some of the answers to these questions.
7.1 Background
We have now reached the last chapter of this book. By this point the readers will
hopefully (if I have done my job as author properly) now have an understanding of
the background of 2DLC, its basic principles and applications, and have some idea
where to start with method development, hyphenation and data analysis. So, where
to from here, what is needed to make 2DLC mainstream?
If you want to get a technique used widely then the best way is to make it easy
to use. Future developments should, therefore, focus on supporting method devel-
opment as shown by Peter Schoenmakers’ group at the University of Amsterdam
(Pirok et al. 2018). In particular, better solutions for method development (perhaps
using advanced software solutions) and data analysis are required when extremely
complex samples (such as biological samples) are analysed; for example, compre-
hensive 2D-LC coupled to IMS/QTOF mass spectrometry. Advances in these areas
and wider recognition of the separation power achievable will push 2DLC into more
routine use.
The other main challenge in 2DLC is education, which is part of the reason behind
this book. Analytical scientists need to not only understand the technology but also
when and where to use it. Applying 2DLC when it isn’t needed or without a proper
understanding of potential problems is likely to lead to disappointment. There are a
huge range of excellent publications and free resources that can be accessed with only
https://doi.org/10.1007/978-981-15-6190-0_7
72 7 Conclusions and Future Developments
Fig. 7.1 3D printed 2D chromatogram created by the author and printed at the advanced
manufacturing precinct at RMIT University
a few clicks on a computer. One particularly detailed one is the Agilent Primer on
2DLC which can be accessed online at https://www.agilent.com/cs/library/primers/
public/5991-2359EN.pdf.
One way we might increase education in 2DLC is to think of new ways to display
the chromatograms. The use of 3D printing is quite common for making molecules
for chemistry education (Jones and Spencer 2018). Taking this one stage further;
once we have a 3D image of the chromatogram in the computer it is possible to map
that surface and create a file that can be taken to a 3D printer. Yes, this means it is
possible to 3D print your 2D chromatograms (this also works for 2D NMR data). An
example of this is shown in Fig. 7.1. In this case the sample that was analysed was
ink from a standard biro.
7.2 3DLC
Going further into the future it may also be possible to perform three dimensional LC
where the first two dimensions are time-based and the third is space-based (Wouters
et al. 2015). This requires a conceptual leap. Rather than a standard HPLC one
must imagine some form of 3D block or cube. The sample starts in one corner
7.2 3DLC 73
then moves along one surface, then another, and then another. At the end of the run
each compound in the mixture is somewhere in the 3D structure of the block. If the
chemical properties of the block varied along its length one would effectively have
hundreds or even thousands of mini columns which could be used to conduct all the
differing column chemistries discussed in the methods and hyphenation chapters of
this book.
We would then have to use something like Raman spectroscopy or imaging mass
spectroscopy to make a 3D depiction of the block over time to create a 3D depiction
(or movie) of the separations. A key issue here would be to achieve adequate flow
control and confinement of the analytes to the desired regions. The image would serve
as the detector so fast image capture and processing would also be needed. There is
a lot of existing technology that could be plugged into this sort of system, but one
can see that the geometry of such a setup would get very complex very quickly. I
am not going to pretend I am anywhere near clever enough to understand how it
would all fit together. Luckily I don’t have to as the design and manufacture of such
a system is part of the Separation Technology For A Million Peaks (STAMP) project
(https://www.stamp-uva.eu/ProjectDescription) running at the Van ’t Hoff Institute
for Molecular Sciences at the University of Amsterdam.
7.3 Column Technology
I think development in column technology, particular monolith columns are likely to

help play a role in 2DLC. A standard HPLC column uses packed columns in which
tiny beads of an inert substance, typically a functionalised silica, are packed tightly
together. A monolith column has no beads but instead consist of continuous beds
containing multiple channels. The channels usually possess a defined bimodal pore
structure with macro- and mesopores in the micro- and nanometer range and a high
surface area available for reactivity. The high permeability and porosity of the silica
skeleton, and the resulting low back pressure allow for more flexible flow rates than
conventional particulate columns. The main advantage of the monolithic technology
is the ability to use high flow rates without significant loss of efficiency. Although not
yet widely used outside specific applications, the wider use of monolith technology
could lead to very much faster 2DLC and even nanoscale 2DLC, separations of
complex mixtures in a much shorter timeframe (Kimura et al. 2004). They also
allow for specialist separations such as that of intact, rather than digested, proteins
(Eeltink et al. 2009).
7.4 Miniaturisation
Another area I feel will really drive advances in 2DLC and analytical chemistry
in general will be the miniaturisation of analytical instrumentation. For instance,
back in the 1980s mass spectrometers were the size of a small room and required
74 7 Conclusions and Future Developments
a computer almost as big, as well as highly trained staff to run them, and, as such,
they were the preserve of the few. It wasn’t until mass spectrometers were small and
robust enough to be used routinely in the lab that their use and applications grew.
The new wave of benchtop NMR spectrometers is a great step in the direction of
smaller instruments. Applications of this technique have already started to take off; I
hope this is just the start. Imagine if you had an LC the size of a computer chip or lab
slide), and/or a mass spectrometer robust enough for you to transport in (and perhaps
even power from) your car, and as easy to operate as your smartphone? This will
require developments in chip-based columns and microfluidics to facilitate but the
potential this would have for the analysis of samples from huge numbers of people
could drive some incredible work in the health and medical sciences as well as a
range of other fields.
References
Eeltink S, Dolman S, Detobel F, Desmet G, Swart R, Ursem M (2009) 1 mm ID poly(styrene-

co-divinylbenzene) monolithic columns for high-peak capacity one- and two-dimensional liquid
chromatographic separations of intact proteins. J Sep Sci 32(15–16):2504–2509
Jones OAH, Spencer MJS (2018) A simplified method for the 3D printing of molecular models for
chemical education. J Chem Educ 95(1):88–96
Kimura H, Tanigawa T, Morisaka H, Ikegami T, Hosoya K, Ishizuka N, Minakuchi H, Nakanishi
K, Ueda M, Cabrera K, Tanaka N (2004) Simple 2D-HPLC using a monolithic silica column for
peptide separation. J Sep Sci 27(10–11):897–904
Wouters B, Davydova E, Wouters S, Vivo-Truyols G, Schoenmakers PJ, Eeltink S (2015) Towards
ultra-high peak capacities and peak-production rates using spatial three-dimensional liquid
chromatography. Lab Chip 15(23):4415–4422
Further Reading
I hope at the end of this book you have gained an appreciation of what two-
dimensional liquid chromatograph is and how it can be used. If you want to learn
more, I have provided a list here of what I consider some of the key papers in the
field.
Pirok, B. W. J., et al. (2019). “Recent Developments in Two-Dimensional
Liquid Chromatography: Fundamental Improvements for Practical Applications.”
Analytical Chemistry 91, 240–263.
Groeneveld, G., et al. (2019). “Perspectives on the future of multi-dimensional
platforms.” Faraday Discussions 218, 72–100.
Pirok, B. W. J., et al. (2018). “Optimizing separations in online comprehensive
two-dimensional liquid chromatography.” Journal of Separation Science 41, 68–98.
Venter, P., et al. (2018). “Comprehensive Three-Dimensional LC × LC × Ion
Mobility Spectrometry Separation Combined with High-Resolution MS for the
Analysis of Complex Samples.” Analytical Chemistry 90, 11643–11650.
Stoll, D. R. and P. W. Carr (2017). “Two-Dimensional Liquid Chromatography:
A State of the Art Tutorial.” Analytical Chemistry 89, 519–531.
Gargano, A. F. G., et al. (2016). “Reducing Dilution and Analysis Time in Online
Comprehensive Two-Dimensional Liquid Chromatography by Active Modulation.”
Analytical Chemistry 88, 1785–1793.
Causon, T. J. and S. Hann (2015). “Theoretical evaluation of peak capacity
improvements by use of liquid chromatography combined with drift tube ion
mobility-mass spectrometry.” Journal of Chromatography A 1416, 47–56.
Wouters, B., et al. (2015). “Towards ultra-high peak capacities and peak-
production rates using spatial three-dimensional liquid chromatography.” Lab on
a Chip 15, 4415–4422.
Giddings, J. C. (1995). “Sample dimensionality: a predictor of order-disorder
in component peak distribution in multidimensional separation.” Journal of
Chromatography A 703, 3–15.
Davis, J. M. and J. C. Giddings (1983). “Statistical theory of component overlap
in multicomponent chromatograms.” Analytical Chemistry 55, 418–424.
https://doi.org/10.1007/978-981-15-6190-0

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SPRINGER BRIEFS IN MOLECULAR SCIENCE

More information about this series at http://www.springer.com/series/8898

ISSN 2191-5407 ISSN 2191-5415 (electronic)

Melbourne, Australia Oliver Jones

Oliver Jones is an analytical chemist based at RMIT University in Melbourne,

Abstract Today’s analytical chemists face increasing demands to maximize the

Modern analytical chemistry is heavily reliant on both increasingly advanced analyt-

Fig. 1.2 Schematic diagram of a 2DLC system

Fig. 1.3 Comparison of standard and comprehensive offline two-dimensional LC separation of

Fully automated 2DLC is a relatively new technique currently offered by systems

Abraham MH (2004) 100 years of chromatography—or is it 171? J Chromatogr A 1061(1):113–114

Giddings JC (1995) Sample dimensionality: a predictor of order-disorder in component peak

Abstract Two-dimensional liquid chromatography is not simple but neither is it

Keywords Analytical chemistry · Orthogonality · Peak capacity ·

The “next frontier” is two-dimensional liquid chromatography where the extra

2.2 Column Orthogonality

The fundamental issue of LC × LC is the combination of two different separation

Fig. 2.1 Stylized 2DLC

Fig. 2.2 Stylised 2DLC

2.3 Peak Capacity and Undersampling

PCt = PC1 × PC2

Fig. 2.3 Illustration of the effect of sampling frequency on peak shape

What we see as a continuous peak on the computer screen is in reality a series of

PCt = (PC1 /Cf ) × PC2

2.4 Surface Coverage

PCt = (PC1 /Cf ) × PC2 × fcoverage

There is also a scan delay for non-chemical directors so we could, if we were

PCt = (PC1 × PC2 × fcoverage )/Cf

2.5 Gradient Elution Chromatography

2.6 Solvent Considerations

2.7 Column Temperature

In gas chromatography separations the temperature program used is an essential

2.8 How to Use 2DLC

Abstract Two-dimensional liquid chromatography offers a way to greatly increase

Keywords Columns · Computability · Modulation · Offline · Online · Separation

3.2 What Factors Should I Consider?

3.3 Offline Method Development

3.4 Online Method Development

An online 2DLC method can be complicated to develop and involves compromises

3.5 Column Selection (Orthogonality)

In gas chromatography (GC) the mobile phase is specifically chosen to be inert to

Pandohee J, Stevenson P, Zhou X-R, Spencer M, Jones O (2015) Multi-dimensional liquid

Keywords Algorithm · Chemometrics · Deconvolution · In silico · Processing ·

4.2 Displaying the Data

4.3 Algorithms and Processing Methods for 2DLC Data

When peak information is in hand, chemometric processes can also applied to

4.4 In Silico Method Optimisation

in silico optimisation of LC separations (Bradbury et al. 2015). Although not as

Abstract The identification of unknown compounds is of fundamental importance

Keywords Collision cross section · Ion chromatography · Electrophoresis ·

5.2 Mass Spectrometry

comes in almost as many varieties as chromatography. Which is best? Well, resolving

spectrometry (IMS–MS) are increasingly being used to address challenges associated

mixtures of compounds. Non-targeted analysis of a wastewater sample with a four-

5.3 Multiple Chromatographic Methods

5.4 Combining Chromatography and Electrophoresis

Multidimensional electrophoretic and chromatographic separations can be powerful

The linking of an LC to a GC system was first published by Majors in 1980, who

Recent developments in modulation technology and automated computational

Donato P, Cacciola F, Sommella E, Fanali C, Dugo L, Dachà M, Campiglia P, Novellino E, Dugo

Wei Y, Lan T, Tang T, Zhang L, Wang F, Li T, Du Y, Zhang W (2009) A comprehensive two-

Abstract It has been shown that two-dimensional liquid chromatography offers a

Keyword Forensics · Natural products · Lipids · Pharmaceuticals · Polymers ·