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Current Analytical Chemistry, 2021, 17, 339-354

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Current
Analytical
Multiple Heart-Cutting Two-Dimensional Liquid Chromatography: Chemistry

Recent Developments and Applications

Brynish R. Dsouza1, Alex Joseph1,*, Subham Das1 and Angel T. Alex2

1
Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher
Education, Manipal, Karnataka, India; 2Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceu-
tical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India

Abstract: Background: Over the last few decades, there has been a growing interest in Two-
Dimensional Liquid Chromatography (2D-LC). This advanced technology has become a relevant so-
lution for solving all the analytical challenges encountered in the pharmaceutical industry. The pri-
mary purpose of this review is to give a brief account of the developing techniques notable in 2D-LC.
Methods: Review of recent literature on advanced separation techniques for complex mixtures re-
vealed that 2D-LC is the most widely used technique in the pharmaceutical laboratory for the separa-
tion of complex samples. Among the various techniques used in 2D-LC, comprehensive 2D-LC and
ARTICLE HISTORY
heart-cutting 2D-LC are highly used techniques. To enhance the resolution, further development has
been made to heart-cutting 2D-LC called multiple heart-cutting 2D-LC, which has recently become a
Received: March 06, 2020 very popular technique in the pharmaceutical industry. Therefore research articles mentioning the use
Revised: May 06, 2020 of multiple heart-cutting 2D-LC for separation of complex mixtures were reviewed.
Accepted: May 29, 2020

DOI: Results: The most crucial benefit of multiple heart-cutting 2D-LC is its reduced effort in method de-
10.2174/1573411016666200630114826 velopment and also the storage of multiple sample sections for further analysis. The multiple heart-
Current Analytical Chemistry

cutting 2D-LC is suited, preferably for method development and impurity analysis of pharmaceutical
substances, fine chemicals and offers better peak separation, which is otherwise not possible by com-
prehensive 2D-LC.
Conclusion: The advantage of two-dimensional liquid chromatography over one-dimensional liquid
chromatography is its high resolving power. Further improvement in resolution was achieved by us-
ing the advanced multiple heart-cutting 2D-LC which appears to have an imperative role in pharma-
ceutical and food analysis.

Keywords: Analytical, heart-cutting, liquid chromatography, one-dimensional, pharmaceutical, two-dimensional.

1. INTRODUCTION tiny packing particles and a comparatively high pressure for

forcing liquid mobile phase through this column is referred
Chromatography is a technique used for the separation of to as high-performance liquid chromatography (HPLC) [1-
a mixture. In Chromatography, two phases are typically pre- 5]. 2D-liquid chromatography is a successful analytical sepa-
sent, one is the mobile phase, and the other one is the sta- ration tool in which the injected sample is separated in two
tionary phase. In analytical chromatography, the analyte sep- different stages by passing through two different chromato-
arated during the chromatography process is identified and graphic columns connected in sequence [6].
its probable concentration is determined in a sample. In liq-
The 2D-liquid chromatography technique is highly pre-
uid chromatography (LC), the liquid is used as a mobile
ferred over conventional one-dimensional liquid chromatog-
phase for separation which is allowed to pass through a col-
raphy due to its high interest in the separation of complex
umn with a stationary phase made up of irregularly or spher-
ically shaped particles. Modern liquid chromatography that samples [7, 8]. One of the most significant advantages of
commonly uses a stationary phase comprising of a column of 2D-LC, when compared to 1D-LC, is that it gives well-
increased peak capacity [9].
An instrument of 2D-liquid chromatography consists of
*Address correspondence to this author at the Department of Pharmaceuti-
cal Chemistry, Manipal College of Pharmaceutical Sciences, Manipal Acad- two HPLC systems named the first dimension (1D) and se-
emy of Higher Education, Manipal, Karnataka, India; Tel: 09448548060; cond dimension (2D), which in turn are connected by the
E-mail: [email protected] loop and a valve system. In this technique, the samples in-
1875-6727/21 $65.00+.00 © 2021 Bentham Science Publishers
340 Current Analytical Chemistry, 2021, Vol. 17, No. 3 Dsouza et al.

Fig. (1). Concepts of 2D-LC. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

jected get separated through two different separating stages. Another example is that this method is also used to select
In 2D-LC (Fig. 1), pump 1 generates the first dimension gra- clone for monoclonal antibody [21]. The goal of 2D-LC
dient. The auto-sampler injects the sample and separation is heart-cutting is somewhat different from that of comprehen-
done by column 1; the 2D-LC injector connects the first di- sive chromatography in that it seeks to evaluate only one or a
mension to the second dimension and thus stores the sample few compounds, characteristic of high-complexity samples.
peaks. Then these stored peaks are re-injected in the second
Therefore, for 2D re-analysis, only a distinct fraction of
dimension, separated by a second column and measured by
the 1D-peak is taken. This breaks the connection between the
detector 2. The LC×LC systems are more prevalent in the
sampling time and the 2D cycle so that both dimensions can
analytical field; thus, this commercially available method has
operate under more optimal conditions, allowing the use of
also entered the UHPLC market and laid its mark [10, 11]. longer gradients and columns with higher 2D separation effi-
There are two types of 2D-LC techniques, the compre- ciency, which often leads to better chromatography com-
hensive 2D-LC and heart-cutting 2D-LC [12]. In compre- pared to comprehensive mode [22].
hensive LC×LC (Fig. 2A), the entire effluent is transferred
Due to this advantage, further development was made to
from the first dimension to the second dimension, and thus
heart-cutting 2D-LC called multiple heart-cutting 2D-LC
the samples get separated in both the dimensions [13]. On
(Fig. 2B). This technique allows the cutting of various mul-
the other hand, in the heart-cutting method, the selected sec-
tiple sections from the first dimension. Each section is stored
tions from the first dimension cut are analyzed in the second
in a deck system until the second dimension is ready for
dimension. The second dimension run time is independent of
analysis. In multiple heart-cutting 2D-LC, a valve connects
the first dimension sampling time; therefore, it gives other
two HPLC systems and that valve allows to cut and store up
chromatographic resolution when compared to one dimen-
to 12 cuts at a time, and thus, these cuts are further analyzed
sion liquid chromatography [14]. In a comprehensive meth-
in the 2D [23]. In multiple heart-cutting 2D-LC, the increased
od, the second dimension run time is limited to the first di-
resolution is mainly used to separate the specific analytes
mension sampling time. Thus, to avoid the loss of separation
from complex samples to distinguish those further and allow
obtained from 1D, fast sampling times are required. When
quantification of low-level impurities. The quantification
both the techniques are compared, the comprehensive tech-
takes place by using tandem mass spectrometry coupled with
nique takes a longer duration of time than the heart-cutting
2D-LC. Therefore, this drawback led to increased popularity 2D-LC [24]. The resolution also checks or evaluates the
of heart-cutting 2D-liquid chromatography in all the labora- specificity of the method by investigating the main peak pu-
tories [15, 16]. rity [25]. The combination of achiral first dimension with
second dimension ultrafast chiral reverse phase method also
Heart-cutting 2D-liquid chromatography is mainly used is used for MHC 2D-LC [26]. One of the main advantages of
to analyze one or more components that are present mostly MHC is that the second dimension of MHC 2D-LC is useful
in complex samples. This method is considered one of the to remove the analytes from the highly water-soluble buffer
widely used techniques for impurities analysis in QC labora- and salts that are required for first dimension separation
tories [17]. The method validation of this technique showed mainly while analyzing biopharmaceuticals. The chromato-
perfect accuracy, robustness, and sensitivity [18]. The heart- graphic systems with MS incompatible buffers or very high
cutting 2D-liquid chromatography consists of two methods salt concentrations are necessary for the analysis of bio-
named single heart-cutting 2D-LC and multiple heart-cutting pharmaceuticals [27].
2D-LC methods. The single heart-cutting technique is ade-
quate to resolve the co-elution of critical components; for Multiple heart-cutting 2D-LC is useful to analyze the
example, the quantitative determination of nicotine in tobac- components that co-elute in 1D separation. In this method,
co leaves. The above application used the tandem mass spec- the first dimension fractions are stored in loops and analyzed
troscopy as a tool for its detection [19]. Further, in 2D-LC, in the second dimension. The main advantage of MHC 2D-
the combination of heart-cutting with switching column is LC over comprehensive 2D-liquid chromatography is the
used to analyze simultaneous metabolomics and lipidomics decoupling of 2D and 1D. In MHC 2D-LC, the analysis time
[20]. of the second dimension is no longer forced to match the
Multiple Heart-Cutting Two-Dimensional Liquid Chromatography Current Analytical Chemistry, 2021, Vol. 17, No. 3 341

Fig. (2). Modern interfaces for (A) Comprehensive 2D-LC. (B) Multiple heart-cutting interfaces with two parking decks A and B bearing six
sampling loops. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

modulation frequency, thus enabling slower and better- liquid chromatography, and how it is used successfully for
optimized two-dimensional runs [28]. Besides, MHC 2D-LC analysis of impurities, employing different modulation tech-
is coupled with 2D-LC ion mobility TOF MS in a low- niques, recent advancements and various applications.
resolution mode for investigation of Ginkgo plant extract.
This low-resolution mode creates a single peak for MS that 2. MULTIPLE HEART-CUTTING TWO-DIMENSIONAL
accelerates structural analysis [29]. In MHC 2D-LC, the re- LIQUID CHROMATOGRAPHY-AN OVERVIEW
moval of heart-cuts can be done at high frequency from vari-
This section gives a detailed account of multiple heart-
ous multiple first dimension peaks, which provide detailed
cutting 2D-liquid chromatography. The MHC 2D-liquid
information on target components in complex samples [13].
chromatography with higher resolution is used to separate
The concept of multiple heart-cutting 2D-LC studies is the specific analytes from most complex samples and thus
mainly to advance the heart-cutting analysis to a higher lev- characterize these analytes, which allows quantification of
el. Innovative interfacing technology enables 1D aliquots to low level of impurities using mass spectroscopy coupled
park while running 2D cycles. Through this way, at high with 2D-LC [31]. This increased resolution helps to investi-
frequency, heart-cuts can be taken from several 1D peaks, gate the main peak purity to examine the specificity of the
providing precise information about target components [23]. method [32]. Recent explorations of MHC 2D-LC suggest
In addition, the use of 2D gradients and column in MHC 2D- using more friendly software so that this technique can be-
LC with higher separation efficiency often leads to improv- come a routine application in the analysis field to analyze a
ing chromatography compared to comprehensive mode, and sample that represents a pharmaceutical compound. In this
therefore it is not necessary to reduce the 1D flow rate to method, each peak is detected at first dimension subjected to
deficient levels as in another mode [30]. The purpose of this heart-cutting, and these peaks are analyzed in the second
review article is to provide information regarding the con- dimension separation using different selectivities to discover
cept of multiple heart-cutting technique in two-dimensional the co-eluting peaks in the first dimension separation [33, 34].
342 Current Analytical Chemistry, 2021, Vol. 17, No. 3 Dsouza et al.

Fig. (3). Illustration of multiple heart-cutting 2D-liquid chromatography. (A higher resolution / colour version of this figure is available in the
electronic copy of the article).

Fig. (4). Detection of peaks in 1D and 2D standard heart-cutting technique. (A higher resolution / colour version of this figure is available in
the electronic copy of the article).

Multiple heart-cutting techniques cooperate with 2D- This problem can be overcome by using a setup called
liquid chromatography to separate the specific analyte from multiple heart-cutting 2D-liquid chromatography. Here (Fig.
most complex samples [35]. This article describes the multi- 5), the sampling loops from the valve of 2D-liquid chroma-
ple heart-cutting (MHC) 2D-LC, a technique that prevents tography are interchanged with 6-position/ 14-port valves,
the loss of relevant information by allowing the parking of each provided with six loops. In this technique, the co-
additional cuts while running the 2D-cycle. The heart-cuts eluting peaks are cut out and stored in the loops, followed by
are removed from first dimensional peaks, and thus the sam- analysis as soon as the second dimension is free [40].
pling permits the in-depth analysis of the green peak in the
second dimension (Fig. 3). The configuration of sampling
loops is shown in Fig. (2B); here the (parking) decks are
placed by replacing the sampling loops of a dual-loop inter-
face, each containing six loops, and therefore in total, the
MHC interface consists of 12 sampling points [36].
In the standard heart-cutting technique, the second di-
mension gradient time is much longer than the comprehen-
sive technique [37]. The main disadvantage of the standard
heart-cutting technique is that peaks cannot be analyzed
while the second dimension gradient is still running [38]. For
example, as shown above (Fig. 4), the first peak (red) is ana-
lyzed by the gradient second dimension, while the second
(purple) and third peaks (blue) elute from the 1D column.
When the 2D is ready, the 4th peak (yellow) can be analyzed
that elutes from the first dimension; thus, the 5th peak (cobalt Fig. (5). Multiple heart-cutting interfaces. (A higher resolution /
blue) cannot be analyzed while the second dimension is oc- colour version of this figure is available in the electronic copy of
cupied [39]. the article).
Multiple Heart-Cutting Two-Dimensional Liquid Chromatography Current Analytical Chemistry, 2021, Vol. 17, No. 3 343

Fig. (6). Detection of peaks in 1D and 2D multiple heart-cutting techniques. (A higher resolution / colour version of this figure is available in
the electronic copy of the article).

Fig. (7). Illustrates the Second dimension solvent gradient composition. (A higher resolution / colour version of this figure is available in the
electronic copy of the article).

Fig. (8). Illustrates Shift in second dimension gradient composition. (A higher resolution / colour version of this figure is available in the
electronic copy of the article).

Here the peaks that cut out and stored during a run are In the above case (Fig. 8), the second dimension gradient
analyzed in the 2D while the 1D is still running. Thus these composition has shifted to match the change in the solvent
peaks are analyzed in the reverse order of storage in single composition of the first dimension.
multiple heart-cutting valves in order to avoid the carry-over
Multiple heart-cutting experiments are usually performed
during the analysis (Fig. 6) [41].
in two operational modes, i.e. first peak-base followed by time-
2.1. Gradient Shift base. In peak-base mode, upon 1D peak recognition, the heart
cuts are taken automatically for 2D analysis. When the first
When the first dimension requires a gradient elution for dimension detector senses the peak, the system is activated
the chromatographic separation, this makes it necessary to for sampling, and the trigger for this can be a combination of
change the solvent gradient in the second dimension along both slope and baseline threshold or single slope and single
with inducing a change in the solvent composition of the first baseline threshold. When the trigger matches the second
dimension. The gradient shift that sets up in the second di- point, the sampling stops or the sampling time gets elapsed.
mension gradient timetable handles these changes [42]. The software manages the delay between first dimension
In the above case (Fig. 7), there is no change in the 2D peak detection and its arrival at the sampling loop inlet [43].
(second dimension) solvent gradient despite the difference in On the other hand, in time-base mode, the time events
the 1D (first dimension) solvent composition. designated by the operator trigger the samples. The software
344 Current Analytical Chemistry, 2021, Vol. 17, No. 3 Dsouza et al.

based on the uploaded chromatogram using peak base mode the first dimension is sampled into one of the sampling
criteria automatically generates the heart cut timetable. Thus, decks. Once the valve switches, the content from the deck is
the 2D-LC viewer assists the data [44]. injected into the column of the second dimension, whereas
the other deck is connected to the first dimension separation
2D-LC facilitates the MHC method setup and data analy-
(Fig. 9) [52].
sis. This method is used for the detailed examination of mul-
tiple target compounds. It consists of unique features that
help to check the repeatability and correctness of the results.
Due to this improved technology, this method provides better
quantification performance, which shows that the MHC 2D-
liquid chromatography is frequently used for method devel-
opment and analysis of impurities present in more complex
samples or fine chemicals [45, 46].

2.2. Modulation
In MHC 2D-LC, one of the significant drawbacks in
method development can be the incompatibility between
both dimension solvent systems (1D&2D). This drawback
can be solved by incorporating different modulation tech-
niques [47].
The heart of every MHC 2D-liquid chromatography is Fig. (9). Passive modulation for multiple heart-cutting 2D-liquid
the modulation interface that plays the role of shifting frac- chromatography. (A higher resolution / colour version of this figure
tions of effluent from 1D to 2D column. Here the modulator is available in the electronic copy of the article).
is the interface between two chromatographic dimensions
[48]. The most commonly used tool for transferring fraction In the above modulation (Fig. 9), the effluent from 1D is
is the 10-port valve or 8-port duo valve. In addition to this, sampled into one deck, and the contents from the other deck
some research activities are in progress in search of alterna- are introduced into the 2D [53]. The process of the decks
tive modulation strategies [49]. filled in one direction and emptied in another direction is
also called a “backflush” mode. In MHC 2D-LC, the decks
Different types of modulation are incorporated in multi-
containing an array of sampling loops are placed to permit
ple heart-cutting 2D-LC. Generally, there are two categories
the storage of more than one fraction [54].
of modulation:
Limitations of Passive Modulation:
• Passive modulation and
i. The volume in the sampling deck should not exceed
• Active solvent modulation.
the limit to prevent the overloading of the sample in
In passive modulation, the effluent from 1D shifted to the the second dimension column [55].
following separation column (2D) is unmodified. In active ii. Secondly, the sampling deck cannot be too small to
solvent modulation, the parts of the effluent from 1D are analyze only a few quantities of effluent from the
modified before entering the subsequent separation dimen- first dimension that has to sample.
sion (2D). Apart from these two, the stationary-phase assisted
modulation and vacuum evaporated modulation have been iii. The frequent switching of the modulation valve re-
introduced under MHC 2D-liquid chromatography [50]. duces the 2D lifetime [56].
To overcome this incompatibility issue, some modifica-
2.2.1. Passive Modulation
tions were made to the modulation process called active sol-
So far, passive diffusion is one of the common forms of vent modulation. In this modulation, chromatographers make
modulation in MHC 2D-LC. In passive modulation, a 10- an effort to alter the 1D effluent matrix to intercept the in-
port or an 8-port duo-switching valve is present, and these compatibility issues and mainly focus on improving 2D sepa-
valves connect the separation dimensions using two or more ration rather than affecting it negatively [57, 58]. Table 1
sampling decks [51]. This modulation strategy is straight- shows examples for the usage of passive modulation in
forward and effective. In this modulation, the effluent from MHC 2D-LC.

Table 1. Examples of passive modulation.

S. No. Passive Modulation

1 Implementation of two identical cyano trap columns for improvement of the RPLC × RPLC-MS separation of steroids in bovine urine [59].

2 Passive diffusion has been implemented for the separation of polyphenols using trapping columns by combined HILIC and RPLC method [60].
Multiple Heart-Cutting Two-Dimensional Liquid Chromatography Current Analytical Chemistry, 2021, Vol. 17, No. 3 345

Fig. (10). Illustration of functions of active solvent modulation in multiple heart-cutting 2D-liquid chromatography interface, including a
new ASM valve, which incorporates an ASM capillary. (A higher resolution / colour version of this figure is available in the electronic copy
of the article).

2.2.2. Active Solvent Modulation concentration before the injection into the 2D [66]. Stoll and
co-workers also introduced multiple numerical methods to
The active modulation was introduced in 2017 [61]. This
predict the effects of active solvent modulation parameters
modulation was mainly introduced to increase the solvent
like dilution factor, injection volume, retention time, and
compatibility for both separation dimensions. Sometimes the
peak width [67]. Various examples demonstrating the appli-
harmony between the first and second dimension mobile
cation of active solvent modulation in MHC 2D-LC are
phase is an issue because the 1D effluent (methanol, acetoni-
summarized in Table 2 [68, 69].
trile, and tetrahydrofuran) can cause peak broadening, distor-
tion, and sample breakthrough in 2D [62]. To overcome these Advantages of Active solvent modulation.
problems, active modulation is done by trapping dilutions
from 1D eluent with weaker mobile phase on the solid-phase i. Dilutes possess an incompatible solvent
extraction cartridges. Thus elevated deck fillings are used to ii. Robust and relatively simple implementation
shorten the run times [63].
iii. On-column focusing on the second dimension
The valve has four positions. Position A and C: 1D efflu-
ent is sampled into deck A & deck B while 2D cycles run Disadvantages of Active solvent modulation.
through other decks. Position B and D: 2D path deck is
placed parallel to ASM capillary [64]. i. Mainly used for 2D reversed-phase.

In active solvent modulation (Fig. 10), the two ports ii. Modulation volume is still a limiting factor [68].
along with a bypass capillary are added to a typical valve and
two rotational parts are attached to the conventional valve. 2.2.3. Stationary-Phase-Assisted Modulation
The positions A & C (Fig. 10) show that the effluent from 1D The stationary-phase-assisted modulation was introduced
travels across one of the deck and all 2D mobile phase travels in 2015 (focusing on modulation) [70]. This modulation is
across the other deck, by replacing the formerly collected 1D one of the well-approved active modulation strategies that
effluent into a 2D column. The positions B&D show that the depend on the low volume enrichment columns called
2
D pump flow is cut into two parts, one that passes along the “traps” rather than using large storage decks [71]. Here the
deck, and the other bypasses the deck such that the eluent traps are nothing but the guard columns that contain a sta-
from 2D acts as a diluent for a fraction of 1D effluent [65]. tionary phase similar to that of a 2D column. To enable the
However, active solvent modulation may also result in retention of traps, the 1D effluent is diluted using weak elu-
enhancing the detection sensitivity by analyzing the analyte ent. Moreover, during method development, the waste lines
346 Current Analytical Chemistry, 2021, Vol. 17, No. 3 Dsouza et al.

Table 2. Examples of active solvent modulation.

S. No. Active Solvent Modulation

Reducing dilution and analysis time in online comprehensive two-dimensional liquid chromatography by active modulation is demonstrated using a
1 combination of hydrophilic interaction and reversed-phase liquid chromatography for the separation of tristyrylphenol ethoxylate phosphate surfac-
tants [49].

2 Two-dimensional liquid chromatography with active solvent modulation for studying monomer incorporation in copolymer dispersants [69].

ASM was used to effectively couple HILIC and RP separations of proteins in an online LC × LC format, which is otherwise quite difficult because of
3
the solvent-strength mismatch between the conditions typically used for these two separation modes [64].

ASM 2D-Liquid chromatography was used for the quantitative determination of target molecules in polymer matrixes. ASM was applied in a com-
4 prehensive 2D-LC (SECxLC) mode for characterization of molecular weight and chemical composition distribution of a polymer blend consisting of
epoxy novolac and phenol novolac [62].

The separation of water and fat-soluble vitamins by sLC × LC with the help of HILIC and RP separations using 2D-LC with active solvent modula-
5
tion [68].

Fig. (11). Illustrates the function of stationary-phase modulation for multiple heart-cutting 2D-LC. (A higher resolution / colour version of
this figure is available in the electronic copy of the article).

are fitted with a detector for monitoring the premature elu- Disadvantages of Stationary-phase assisted modulation.
tion from the traps [72]. i. Robustness of the trapping units
The above setup (Fig. 11) demonstrates that the 2D efflu-
ii. Additional complexity to method development
ent is analyzed by the modulator and predicts that the sta-
tionary phase will help in the retention of analytes in the iii. Premature elution of the analytes from the traps
traps. Whereas the first dimension solvent remains unre- [76-78].
tained and thus leaves the system. When the valve switches,
the second dimension gradient program (mobile phase) 2.2.4. Vacuum-Evaporated Modulation
elutes the trapped analytes and introduces these analytes into The vacuum-evaporated modulation was mainly set up to
the second dimension column as sharp, concentrated bands.
overcome the incompatibility issues that arise during the
In SPAM, the mixer inserted optionally enables the retention
merging of normal-phase liquid chromatography and re-
of the traps; this is done by lowering the elution strength of
1 verse-phase liquid chromatography [79]. The central concept
D effluent before entering the traps. The effectiveness of
of VEM is to evaporate the organic effluent from the first
SPAM can be further enhanced by (1) reducing trap vol-
dimension normal phase separation. The storage decks are
umes, (2) increasing traps robustness, and (3) developing
equipped with the heating wires to heat the solvent by facili-
micromixers to fuse the two solvent systems [72, 73]. Table
tating this process. In the end, a vacuum is placed at the
3 shows examples of SPAM in MHC 2D-LC.
modulation loop to aid evaporation.
Advantages of Stationary-phase-assisted modulation. In the above modulation, the 1D effluent passes through a
i. Reduce solvent incompatibility issues heated deck and travels to a vacuum outlet (Fig. 12, left,
blue). Because of elevated temperature and reduced pressure,
ii. Removal of most of the 1D mobile phase [74]
the strong NPLC solvents are expected to evaporate rapidly,
iii. Improvement in detection sensitivity [59]. and thus the analytes are assumed to deposit on the walls of
iv. Decoupling of first and second dimension analysis the deck. Upon switching (Fig. 12, right, red), the eluent
time [75] from 2D is injected into the heating deck and, with the help
of heating wire, rapidly re-dissolves the analytes and intro-
v. Reduced second dimension injection volume [49] duces these analytes to the 2D column [80]. Various exam-
Multiple Heart-Cutting Two-Dimensional Liquid Chromatography Current Analytical Chemistry, 2021, Vol. 17, No. 3 347

Table 3. Examples of stationary-phase assisted modulation.

S. No. Stationary-Phase Assisted Modulation

Recent Implementation of SPAM in an LC × LC workflow for the SCX × RPLC−HRMS characterization of peptides applied to Proteome Analysis
1
of Saccharomyces Cerevisiae [70].

2 SPAM has been implemented in the heart-cut SCX-RPLC-MS/MS separation of tobacco-specific N-nitrosamines in cigarette smoke [19].

3 Implementation of SPAM in the HILIC × RPLC separation of procyanidins from green cocoa beans [72].

4 The recent development of a nanoflow LC × LC system SPAM for the separation of intact proteins [78].

Table 4. Examples of vacuum evaporation modulation.

S. No. Vacuum Evaporation Modulation

A comprehensive 2D-LC (NPLC × RPLC) with a VEM interface was used for the analysis of Traditional Chinese Medicine Radix salviae miltiorrhi-
1
za bage extract [80].

2 Vacuum evaporation modulation is implemented for the analysis of lipidomic profiling in mouse serum [82].

Fig. (12). Illustrates the function of vacuum evaporation modulation used for two-dimensional liquid chromatography. (A higher resolution /
colour version of this figure is available in the electronic copy of the article).

ples summarizing the usage of VEM in MHC 2D-LC are concentrated solution of the substance. The impurities pre-
shown in Table 4. sent in the fine chemicals frequently show structural resem-
blance to the main product, so it is challenging to analyze
Advantages of Vacuum evaporation modulation.
these impurities using a system with a column-solvent com-
i. Incompatibility problems are overcome. bination (different selectivity) [84]. Thus this problem can be
ii. Lipid species analysis in human plasma [81]. resolved by using MHC 2D-LC, as the separation is done by
heart-cutting of co-eluting compounds with different selec-
Limitations of Vacuum evaporation modulation. tivity (column-solvent combination) [85]. In the pharmaceu-
i. The volatility of the to-be-evaporated solvents. tical industry, during the method development, all the impu-
rities must be identified, and accordingly, no impurity should
ii. The extent of loss of volatile solvents. be detected due to co-elution with other compounds or an-
iii. The rate of dissolution for large analytes [82]. other impurity [86]. Therefore, this can be achieved by the
application of multiple heart-cutting 2D-liquid chromatog-
raphy. This technique enables the heart-cutting of multiple
3. MULTIPLE HEART-CUTTING TECHNIQUES FOR peaks from 1D separation, storing these cuts in decks and
IMPURITY ANALYSIS subsequent 2D analysis of the stored heart cuts [87]. The
In the pharmaceutical and chemical industry, the analysis MHC 2D-LC is mainly useful for the analysis of a sample
of impurities is of great importance. According to ICH representing a pharmaceutical compound [88]. In this tech-
guideline Q3A (R2), the contaminants present above 0.05% nique, each peak is perceived after 1D separation is subjected
must be outlined, and impurities above 0.1% should be iden- to heart-cutting, and analyzing these peaks using different
tified [83]. Liquid chromatography plays a vital role in ana- selectivities in 2D separation permits the discovery of possi-
lyzing the impurities in pharmaceuticals products by taking a ble co-elution from the1D [89].
348 Current Analytical Chemistry, 2021, Vol. 17, No. 3 Dsouza et al.

Fig. (13). Removal of interference by MHC 2D-LC. (A higher resolution / colour version of this figure is available in the electronic copy of
the article).

3.1. For Impurity Analysis in the Presence of Severe and the excipients and active pharmaceutical ingredient dom-
Interference inated the mass spectra extracted in the proper retention
time. Therefore the MHC 2D-LC-MS was introduced to
The following example illustrates the ability of MHC
avoid the MS saturation caused by the excipients and the
2D-LC to analyze impurities in the presence of severe inter-
API by heart-cutting of the impurity for 2D chromatographic
ferences. A new impurity was observed in the topical drug
separation to get a pure mass spectrum [94]. From the 2D
product of 0.1%, eluting on the tail of the peak from the ac-
separation, the UV chromatograms showed the presence of
tive pharmaceutical ingredient [90]. During the UV chroma-
both the API and impurity in two cuts [95, 96]. The peaks
togram, the impurity barely separated from the API peak,
separated, and the UV spectra confirmed the elution order of
and the UV spectrum from the impurity was different from
the compound. By using additional dimensions, the amount
the API. Thus, it was not possible to get a better 1D separa-
of interference considerably reduction was observed from the
tion between the API and the impurity [91]. Further, due to
the saturation of MS, a significant tailing of the API MS sig- excipients and API with the impurity [97]. At the retention
nal was observed with mass spectroscopy detection [92]. time of API and impurity, a compound search was per-
formed. With the same mass and isotopic pattern, two iso-
Additionally, over a large part of the chromatogram, huge
meric compounds were found; one corresponding to the API
signals were found from the excipients of the drug product.
and the other the impurity, which could not be established
Thus, the diagram below illustrates this troublesome case
with LC-MS alone [98, 99].
(Fig. 13).
In the above setup (Fig. 13), the six chromatograms of a 4. RECENT ADVANCEMENT IN MHC 2D-LC
sample are analyzed by LC-MS (left) and MHC 2D-LC
(right). The impurity and the active pharmaceutical ingredi- Multiple heart-cutting two-dimensional liquid chroma-
ent eluted near the time segments are marked with the black tography has recently become more popular in the analytical
box. The LC-MS in the left top shows UV chromatograms at world. It is widely used in the separation of samples contain-
280nm, and the middle shows MS total ion chromatograms ing complex mixtures of components to give good chroma-
(TIC) dominated by eluting excipients and the bottom shows tographic performances. Due to this advantage in recent
MS compound chromatograms which only the API detected. years, multiple heart-cutting techniques have reached nu-
The MHC 2D-LC in the right top shows UV chromatograms merous heights in the analytical world. Now the advance-
overlay of two cuts across the impurity, the middle shows ment of this technique has reached the study of toxicity as-
sessment for resolving all isomeric plant metabolites. For
MS total ion chromatograms (TIC) with minimal signals
example, Multiple Heart-Cutting Two-Dimensional Liquid
from the excipients and bottom shows MS compound chro-
Chromatography Quadrupole Time-Of-Flight Mass Spec-
matogram of both corresponding API and impurity [17, 93].
trometry method incorporated to determine isomeric pyrrol-
MS software algorithms were not able to identify the izidine alkaloids and corresponding N-oxides present in food
compound at any appropriate retention time of the impurity, products [24].
Multiple Heart-Cutting Two-Dimensional Liquid Chromatography Current Analytical Chemistry, 2021, Vol. 17, No. 3 349

Further, to separate critical peaks for more accurate tion of relevant impurities in biopharmaceuticals. Further-
measurements, a Multiple-Heart-cutting Agilent 1290 Infini- more, MS classification of impurities is typically achieved
ty 2D-LC Solution with multiple 2D gradients is set up to by collecting offline fractions which, apart from being time-
ensure the best separation at the essential parts of the chro- consuming, often suffer from poor recovery or impurity deg-
matogram for the chromatographic analysis of synthesis radation. The recent development of multiple heart-cutting
products. To develop sophisticated methods more easily than two-dimensional liquid chromatography has provided a way
those existing, Easy-To-Use 2D-LC Software was enhanced to address these problems (Table 5) [28].
to enable the most straightforward setup of MHC methods.
Now all the information regarding 2D-LC can be seen at a 5.3. A Combination of Multiple Heart-cutting 2D-LC and
glance. Even better, the upload of the reference chromato- Quadrupole Orbitrap High-resolution Mass Spectrosco-
gram during the setup process helps to predict the peak- py was Developed for Simultaneous Determination of the
based or time-based form. Multiple heart-cutting chromato- Aflatoxin B1, B2, G1, G2.
grams can be very challenging, specifically when many The first dimension (1D) of the C18 capillary column
heart-cuts have to be obtained and multiple 2D-LC studies was used to separate aflatoxins from the complex matrixes.
have to be carried out. Therefore, to overcome this difficulty, A 2-position/10-port high-pressure valve fitted with two 60
a whole New Heart-Cut Viewer was developed for analyzing μL lops was then used to move the 1D-LC heart-cutting into
all the chromatograms produced in multiple heart-cutting a second-dimensional (2D) separation pentafluorophenyl
studies. It can capture and display a complete 1D chromato- (PFB) column. With the better separation of the MHC-LC /
gram, a table with all the heart-cuts showing their 1D reten- LC system, the method's sensitivity has been improved,
tion period, and an entire 2D chromatogram on a single which is vital for the study of trace mycotoxins (Table 5)
screen for more precise study. The new Agilent 1290 Infinity [101].
2D-LC Solution with Multiple Heart-Cutting provides a
complete Ready-To-Go Solution with all the hardware need- 5.4. Multiple Heart-cutting 2D-LC Widely used in the
ed that allows for the most straightforward system setup with Biopharmaceutical Industry for Various Separation
the New Heart-Cut Viewer for multiple heart-cutting 2D-LC Issues such as Polymer and Peptide Analysis
and unmatched data analysis. This solution makes MHC 2D-
In the field of bioanalysis, three major trends have been
LC even more useful, easier to use, and more relevant for all
identified over the past two decades [30]. Multiple Heart-
analytical practices [36, 100].
Cutting (MHC) 2D-LC is distinct from comprehensive
chromatography in that it attempts to determine only one or a
5. APPLICATIONS OF MHC 2D-LC
few target compounds, characteristic of high-complexity
Multiple heart-cutting two-dimensional liquid chroma- samples [14]. Even before the dramatic growth of biophar-
tography is an advanced technique for the separation of maceutical research, 2D-LC was commonly used in prote-
components from the complex samples. Thus, this technique omics research to distinguish peptides [102]. Now, this prior
is widely applicable to the analysis of various elements pre- knowledge is being applied and extended in the literature as
sent in the pharmaceutical compound; such as: researchers use 2D-LC to detect and understand minor dif-
ferences between therapeutic proteins. Biopharmaceutical
5.1. Loop-based Multiple Heart-cutting Two-dimensional analyzes are extremely complex. It includes a suite of state-
Liquid Chromatography used for Target Analysis in of-the-art analytical methods and instruments with full re-
Complex Matrices such as Additives in Polymers solving power. 2D-LC provides high chromatographic selec-
This loop-based multiple heart-cutting 2D LC is provided tivity and power resolution by integrating complementary
as a solution for quantifying target components in complex separation modes with direct coupling to mass spectrometers
matrixes. Because of peak overlap with polymer compo- (Table 5) [103-106].
nents, the one dimensional LC analysis with UV detection
5.5. A Profiling Method with Quadruple Time-of-flight
did not allow quantitation of isomers. Thus, the MHC 2D-
Mass Spectrometry was Developed in the Study based on
LC analysis provided the separation power, accuracy, and
Multiple heart-cutting, Two-dimensional, Ultra-high Per-
repeatability needed for quantitative analysis of the additives formance Liquid Chromatography
of interest. MHC 2D-LC is, therefore, an effective solution
for quantitative analyses of samples which are difficult to Low molecular weight heparins (LMWHs) are polydis-
distinguish if conventional 1D fails (Table 5) [14]. perse and microheterogeneous polysaccharide mixtures used
as anticoagulant drugs. Analysis of profiling is essential for
5.2. The Multiple Heart-cutting, Two-dimensional Liquid more in-depth insight into the structure of LMWHs. Previous
Chromatography-mass Spectrometry has Recently been oligosaccharide mapping methods are of relatively low reso-
used in Industry to Assess Related Impurities in Bio- lution and cannot show a full picture of the structural nature
pharmaceuticals using Salt-containing Mobile Phases of LMWHs. MHC 2D-LC represents a stable, automated and
"Salty" mobile phases often offer superior chromato- practical approach to profiling LMWHs. Using size-
graphic efficiency but are not consistent with the detection of exclusion chromatography and reversed-phase ion-pairing
mass spectrometry. Peak monitoring is also based on peak chromatography in a two-dimensional separation, LMW
areas during system growth to offer a real-time determina- components of different sizes and LMW components of the
350 Current Analytical Chemistry, 2021, Vol. 17, No. 3 Dsouza et al.

Table 5. Applications of multiple heart-cutting two-dimensional liquid chromatography.

S. No. Applications of MHC 2D-LC

1 The determination of hexabromocyclododecane in polystyrene using loop-based multiple heart-cutting two-dimensional liquid chromatography [14].

The multiple heart-cutting two-dimensional liquid chromatography is used to obtain real-time MS data for bovine insulin-related impurities at a low
2
level [28].

Multiple heart-cutting two-dimensional liquid chromatography is used to obtain high-quality MS spectra for mobile phases containing high concentra-
3
tions of sodium, sulphate and phosphate [28].

Development of multiple heart-cutting two-dimensional liquid chromatography coupled to quadrupole-orbitrap high-resolution mass spectrometry for
4
simultaneous determination of ochratoxin A in snus, a smokeless tobacco product [101].

The MHC 2D-LC method is used to resolve protein and unconjugated small molecule drug as an antibody-drug conjugate material, followed by quan-
5
titation of free drug in a 2D RPLC separation [88].

The MHC 2D-LC is used to rapidly determine which amino acid residues of trastuzumab were deamidated as a result of the pH stress on the protein
6
molecule [105].

Multiple heart-cutting 2D-LC with quadruple time-of-flight mass spectrometry has also been used in the profiling analysis of low molecular weight
7
heparins [104].

Multiple heart-cutting 2D-LC is used for the chiral separation and chemical profile of Dengzhan Shengmai by integrating comprehensive multiple
8
heart-cutting two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry [106].

same size can be solved but with different charges and polar- The MHC 2D-LC permits the heart-cutting of multiple
ities, offering a complete image of an LMWH. Structural peaks from 1D separation and transfers these peaks with dif-
information was then obtained using quadruple time-of-flight ferent selectivities to the 2D separation. The MHC 2D-LC is
mass spectrometry on each portion. More than 80 and 120 suited preferably for method development of impurity analy-
oligosaccharides, respectively, were detected and allocated sis of pharmaceutical substances and fine chemicals. In com-
unambiguously from the LMWHs, nadroparin, and enoxapa- parison with comprehensive 2D-LC, the MHC 2D-LC meth-
rin. This approach may be useful for quality control of od has shown better peak separation.
LMWHs and as an effective tool for glycomics related to
The above articl motivates to increase the development of
heparin (Table 5) [104].
2D-liquid chromatographic methods using MHC 2D-LC.
Thus, multiple heart-cutting 2D-liquid chromatography ap-
CONCLUSION pears to have a bright future in the area of pharmaceutical
Multiple heart-cutting two-dimensional liquid chroma- and food analysis.
tography has been introduced for the separation of samples
containing complex mixtures to give good chromatographic LIST OF ABBREVIATIONS
performances than 1D-LC. This method makes use of inter- 1
D = First Dimension
face technology, which enables the parking of first dimen-
sion aliquots while concurrently running 2D analysis. There- 1D-LC = One Dimensional Liquid Chromatography
fore, these multiple heart-cuts drawn at high frequency from 2
D = Second Dimension
multiple 1D-peaks provide detailed information on target
components present in complex samples. This technique is 2D-LC = Two Dimensional Liquid Chromatography
also useful for the analysis of natural products, pharmaceuti- API = Active Pharmaceutical Ingredient
cals, and polymer samples. The decoupling of 2D from a 1D
ASM = Active Solvent Modulation
time scale allows the possible operation of both dimensions
under optical conditions. Intuitive software shows to assist HILIC = Hydrophilic Interaction Liquid Chromatog-
the MHC 2D-LC operation by method setup and assessment raphy
of 2D information. HPLC = High-Performance Liquid Chromatography
MHC 2D-liquid chromatography is a valuable method for HRMS = High-Resolution Mass Spectrometry
challenging separation problems, including qualitative and
quantitative analyses. It also allows differentiating complex ICH = International Conference On Harmonization
samples which is not possible with the 1D approach. In addi- LC-MS = Liquid Chromatography-Mass Spectroscopy
tion, the structural analysis can be performed efficiently by
incorporating mass spectroscopy to 2D-LC. Thus, this MHC LC×LC = Two Dimensional Liquid Chromatography
2D-LC method reduces the dependence of 2D on 1D and LMWHs = Low Molecular Weight Heparins
hence simplifies method development.
Multiple Heart-Cutting Two-Dimensional Liquid Chromatography Current Analytical Chemistry, 2021, Vol. 17, No. 3 351

MHC = Multiple Heart-cutting [5] Bhattacharyya, S.; Rana, D.; Bhattacharyya, S.N. A Thermodynam-
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Nm = Nanometer ments in two-dimensional liquid chromatography: Fundamental
NPLC = Normal Phase Liquid Chromatography improvements for practical applications. Anal. Chem., 2019, 91(1),
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Q3A (R2) = Impurities in New Drug Substances http://dx.doi.org/10.1021/acs.analchem.8b04841 PMID: 30380827
[8] Davis, J.M.; Stoll, D.R. Likelihood of total resolution in liquid
QC = Quality Control chromatography: evaluation of one-dimensional, comprehensive
two-dimensional, and selective comprehensive two-dimensional
RPLC = Reverse Phase Liquid Chromatography liquid chromatography. J. Chromatogr. A, 2014, 1360, 128-142.
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SCX = Strong Cation Exchange [9] Stoll, D.R.; Carr, P.W. Two-Dimensional liquid chromatography:
sLC = Selective Comprehensive Two Dimensional A state of the art tutorial. Anal. Chem., 2017, 89(1), 519-531.
http://dx.doi.org/10.1021/acs.analchem.6b03506 PMID: 27935671
Liquid Chromatography [10] Filgueira, M.R.; Huang, Y.; Witt, K.; Castells, C.; Carr, P.W. Im-
SPAM = Stationary Phase Assisted Modulation proving peak capacity in fast online comprehensive two-
dimensional liquid chromatography with post-first-dimension flow
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two-dimensional high performance liquid chromatography through
TOF = Time of Flight the use of high temperature ultra-fast gradient elution reversed-
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UHPLC = Ultra-High Performance Liquid Chromatog- 123-137.
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UV = Ultraviolet-visible chromatographic analysis of alcohol ethoxylates. Anal. Chem.,
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S.C. Fast, comprehensive two-dimensional liquid chromatography.
CONSENT FOR PUBLICATION
J. Chromatogr. A, 2007, 1168(1-2), 3-43.
Not applicable. http://dx.doi.org/10.1016/j.chroma.2007.08.054 PMID: 17888443
[14] Pursch, M.; Buckenmaier, S. Loop-based multiple heart-cutting
two-dimensional liquid chromatography for target analysis in com-
FUNDING plex matrices. Anal. Chem., 2015, 87(10), 5310-5317.
http://dx.doi.org/10.1021/acs.analchem.5b00492 PMID: 25897943
None. [15] van der Horst, A.; Schoenmakers, P.J. Comprehensive two-
dimensional liquid chromatography of polymers. J. Chromatogr. A,
CONFLICT OF INTEREST 2003, 1000(1-2), 693-709.
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The authors declare no conflict of interest, financial or PMID: 12877195
[16] Stoll, D.R.; Venkatramani, C.J.; Rutan, S.C. Peak purity in liquid
otherwise.
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ACKNOWLEDGEMENTS [17] Yang, S.H.; Wang, J.; Zhang, K. Validation of a two-dimensional
liquid chromatography method for quality control testing of phar-
One of the authors, Subham Das, is thankful to the Mani- maceutical materials. J. Chromatogr. A, 2017, 1492, 89-97.
pal Academy of Higher Education for providing Dr.T.M.A. http://dx.doi.org/10.1016/j.chroma.2017.02.074 PMID: 28284763
Pai Fellowship. The authors also gratefully acknowledge [18] Sheng, Y.; Zhou, B. High-throughput determination of vancomycin
in human plasma by a cost-effective system of two-dimensional
Manipal College of Pharmaceutical Sciences, for providing liquid chromatography. J. Chromatogr. A, 2017, 1499, 48-56.
facilities for this work. http://dx.doi.org/10.1016/j.chroma.2017.02.061 PMID: 28420531
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