Open access peer-reviewed chapter

Perspective Chapter: Multi-Dimensional Liquid Chromatography - Principles and Applications

Written By

Esayas Tesfaye, Tadele Eticha, Ariaya Hymete and Ayenew Ashenef

Submitted: 12 December 2021 Reviewed: 01 April 2022 Published: 06 July 2022

DOI: 10.5772/intechopen.104767

From the Edited Volume

Analytical Liquid Chromatography - New Perspectives

Edited by Serban C. Moldoveanu and Victor David

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Abstract

Many complex mixtures usually constitute hundreds or even thousands of individual components of interest. Such mixtures are much too complicated to be separated for analytical duties in a reasonable period of time using only a single-dimensional chromatographic method. However, if a complex mixture is separated by an initial dimension using multi-dimensional liquid chromatography, a simpler portion of that separation is collected and goes to the second dimension. Each of these fractions will be analyzed separately, allowing exceedingly complex mixtures to be resolved in a short period of time. This chapter explains the fundamental principles, theoretical discussions as well as various applications with typical examples of multi-dimensional liquid chromatography in different fields.

Keywords

  • multi-dimensional liquid chromatography
  • reversed phase liquid chromatography
  • stationary phase
  • mobile phase
  • chiral separation

1. Introduction

Many complex mixtures consist of hundreds or even thousands of distinct components of interest. Some of these mixtures are so convoluted that they have never been isolated fully and may never be. Obviously, such mixes can be separated unidimensionally to some degree, but there is little possibility that all of the mixture’s components will elute. They will not be separated completely even by exhausting all options of using a highly efficient column under ideal circumstances and altering the chromatographic conditions (such as solvent system composition, column temperature, and mobile phase pH) throughout the elution [1, 2].

The peak capacity of one-dimensional liquid chromatography for the analysis of complicated samples is limited. To resolve as many compounds as feasible, techniques with larger peak capacities are required. The employment of multi-dimensional chromatography could be a feasible solution to this challenge. So, two-dimensional liquid chromatography (2D-LC), which has a long history in a variety of analytical domains such as proteomic and genomic research, could be a useful technique for the thorough study of complex samples [2, 3, 4, 5].

In 2D-LC configurations, a variety of chromatographic methods have been used, with RP being the most popular due to its greater compatibility with electrospray ionization (ESI) MS, high-resolving power, and sample desalting capability options when the first dimension demands salt gradients. Because of the good orthogonality of these two separations, the vast majority of 2D-LC analyses implemented today use Strong Cation Exchange (SCX) coupled to Reverse Phase (RP) in both on-line and off-line modes. Other 2D-LC methods, including as size exclusion chromatography (SEC), affinity purification chromatography (AFC), various types or combinations of ion exchangers, anion and cation mixed-bed exchange, and hydrophilic interaction liquid chromatography (HILIC), have emerged in recent years as promising alternatives to this combination [5, 6, 7, 8].

Giddings in 1984 was the first to establish the theory of multi-dimensional chromatographic separations. The history of “multi-dimensional” liquid separations is almost as long as that of chromatography. The word refers to the method in which a sample is subjected to many separation mechanisms such as mobile phase modifier concentration, mobile phase pH, and column temperature. This is designed considering the physicochemical properties of the sample components and each of which again counted as an independent separation dimension in one step. The resulting 2D system has a higher-resolving power than each single dimension when two separation systems based on different (non-correlative) retention mechanisms are coupled. One-dimensional liquid chromatography is a single-step process using only one column, while multi-dimensional liquid chromatography uses two and more than two steps and columns to separate samples. At the same time, the peak capacity, separation efficiency, sample resolution, and complexity of the method increase when the user opts from one to multi-dimensional liquid chromatography. The most popular version of multi-dimensional chromatography is the two-dimensional liquid chromatography. Early multi-dimensional separations were performed in both planar and columnar modes using only a combination of paper chromatography, electrophoresis, and gels. Chromatographic advancements have boosted separation power in terms of the number of analytes separated, but this has switched focus to the separation of highly complex mixtures such as proteomics and metabolites [3, 4].

In light of its enormous potential, a number of researchers had begun to pioneer the next major step of 2D technology isoelectric-focusing X-gel electrophoresis was evolved by O’Farrell and others into a 2D technology capable of separating over 1000 proteins. Another scientist Guiochon embarked on a project to convert 1D column LC into a 2D column method [9].

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2. Principles of multi-dimensional liquid chromatography

To achieve effective separations in a comprehensive multi-dimensional LC technique, the development and optimization of it necessitates the adjustment of several parameters.

2.1 Column selectivity

When building an MDLC separation, column selectivity critically affects MD system, and orthogonality (independent separation mechanisms) and finally, peak capacity. To get the greatest possible increase in peak capacity, the columns employed in the two dimensions must have varying degrees of selectivity. When optimizing a process, a number of additional variables must be taken into consideration, including mobile phase composition, flow rate, and temperature. Because two-dimensional systems with totally non-correlated selectivities are uncommon in reality, matching and optimizing the operating circumstances in both dimensions are necessary to gain a considerable boost in resolving power [10].

2.2 Orthogonality

When MDLC separation is orthogonal, it means the two separation mechanisms are independent of each other, and provide complementary selectivities. To achieve orthogonal separation, columns employed in MDLC must be different in terms of dimensions and the composition of the stationary phase taking into account the physicochemical properties of the sample components including size and charge, hydrophobicity, and polarity. In 2DLC, the most critical and difficult decision is choosing which columns should be used as the first and second step. This decision affects the system’s separation capabilities. The optimum result is attained when columns retain substances in a distinct way, resulting in a unique separation process. The larger the variation becomes in column chemistry, the higher the effectiveness of the separation process [4, 11, 12].

2.3 Peak capacity

The peak capacity of multi-dimensional separation system is the maximum number of peaks to be separated on a given column. In multi-dimensional chromatography, the peak capacities are multiplicative, which is the best assessment of performance under gradient settings. According to the product rule, in ideal circumstances a 2DLC system’s overall peak capacity is the product of the peak capacities of the two dimensions [13, 14].

2.4 Resolution and sampling rate

Keeping the first-dimensional resolution is a vital criterion to follow in a complete 2D separation, which may be done by conducting a sufficient number of peak samples. To obtain the highest two-dimensional resolution, each separated peak in the first dimension should be sampled at least three times into the second dimension. The sampling time and rate affects the analysis time and its resolution. The shortest sampling time or rate into the second dimension gives the best resolution and longer sampling times decrease resolution. At the same time, the analysis time of the second-dimensional separation system is a major factor in determining the total analysis time of comprehensive two-dimensional separation systems and its resolution [15, 16, 17].

The most critical factors affecting the results of an on-line 2DLC separation are the effects of the stationary phase, mobile phase, and temperature on separation selectivity and peak capacity, compatibility of mobile phase in each dimension, and the matching of column dimensions and flow rates in each dimension. In general, excellent orthogonality across the different dimensions, great peak capacity in each dimension, preserving the early dimensions’ peak capacity, and reducing sample loss throughout the process are all considered to be fundamental principles for a productive multi-dimensional design [4, 15].

2.5 Combinations of separation modes in MDLC

Multi-dimensional liquid-based separation technologies have been constantly improved and innovated to get better results throughout time. MDLC may be used in a variety of ways to maximize its separation power, depending on the analytical application. Among the potential separation mode combinations are ion exchange chromatography/reversed-phase chromatography (IEC/RPC), size exclusion chromatography/reversed-phase chromatography (SEC/RPC), size exclusion chromatography/ion exchange chromatography (SEC/IEC), normal-bonded phase chromatography/reversed-phase chromatography (NPC/RPC), liquid-solid chromatography/reversed-phase chromatography (LSC/RPC), affinity chromatography/reversed phase chromatography (AC/RPC), and achiral column/chiral column. In today’s 2D-HPLC/MS coupling, the most common separation strategy is strong cation exchange (SCX) in the first dimension, followed by RPLC in the second dimension. This is because SCX is the best terms of sample capacity, whereas RPLC is the most compatible column with MS. The various techniques used in multi-dimensional chromatography are described in Table 1 [16, 17, 18].

MDLC ModesFirst dimensionSecond dimensionCharacteristics
SCX-RPLCChargeHydrophobicityOrthogonality, high capacity, and efficiency, most wide application
HILIC-RPLCHydrophilicity/polarityHydrophobicityOrthogonality, good selectivity of PTMs (post-translational modification in proteomics)
SEC-RPLCMolecular sizeHydrophobicityOrthogonality, low-resolving power, and high-loading capacity of SEC for sample fractionation
CIEF-RPLCIsoelectric pointHydrophobicityOrthogonality, strong sample concentration of CIEF
RPLC-RPLCHydrophobicity under different pH conditionsHydrophobicity under different pH conditionsLimited orthogonality, excellent resolving power

Table 1.

Multi-dimensional liquid chromatography combination modes.

CIEF: Capillary Isoelectric focusing; SEC: Size exclusion chromatography.

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3. Construction modes of MDLC

MDLC is often constructed using comprehensive 2DLC and heart-cutting 2DLC. A comprehensive or heart cut onto a different chromatographic column with a greater suited selectivity may redirect co-eluting components into independent eluting components. A divert valve facilitates flows and sampling elute and diverts all or portion of the analyte of interest plus co-eluted compounds from the initial column selectively to the second column for further separation and resolution. The heart-cutting MDLC is utilized to improve component separation. This is done to pre-separate targeted constituents from interfering matrices, and only specified single fractions are sent to the second dimension. It uses typical LC conditions, specifically flow rate, which is ideal for low-abundance component identification and purity analysis [14, 17, 19].

In multiple heart cuttings, more than one area of the 1D effluent is injected onto the 2D column. 2DLC is a good technique to tackle pharmaceutical issues. In pharmaceutical analysis, only certain peaks or sections of the first dimension are of relevance; hence, its eluent does not need to be transferred to the second dimension. On-line multi-heart cutting provides more versatility. In comprehensive MDLC, it is feasible to collect as much information as possible by doing a non-targeted and thorough examination of complicated samples. It utilizes 2DLC, which transfers all fractions from first into second dimension for subsequent analysis. As explained in Table 2 below both heart cutting and comprehensive implementation of multi-dimensional liquid chromatography have its own advantages and disadvantages. One has its own superior field of application over the other [20, 21, 22].

ImplementationNumber of target compoundsAdvantagesDisadvantagesTypical application
Heart cut (LC-LC)+Simple; powerful for highly targeted workLimited to a few target samplesTargeted analysis in complex matrix (e.g., drug metabolite in
serum)
Multiple heart cut
(mLC–LC)
++Amenable to more
target compounds
More complex instrumentationQuantitation of moderately complex mixture (e.g., mixture of achiral/chiral molecules)
Comprehensive (LC × LC)+++Most efficient way to obtain full of sample composition can see several peaks in reasonable timeSlow speed of 2D separation, long analysis timeSample profiling/fingerprinting (e.g., metabolomics), discovery (untargeted) type analysis

Table 2.

Comparison of key attributes of the major implementations of MDLC.

+, ++, +++ denotes an increase in the number and complexity of target compounds of interest for the analytical work.


Multi-dimensional systems may be coupled in three ways: on-line, stop-and-go, and off-line. Multi-dimensional on-line separation allows for direct transfer of fractions from one dimension to the next for further separation. On-line coupling involves coupling the second dimension to the first dimension in real time. Under this separation system, the second analysis of a single fraction should be performed within the time it takes to collect, transport, and analyze the fraction. The key benefits are the lower sample size required, less sample loss, and faster analytical times. It has more strict conditions, such as the first dimension’s solvent must be a weak eluent in the second dimension, and the second dimension must be quick enough to maintain the first dimension’s resolution. Using on-line setups reduces sample handling since sample movement across dimensions is continuous through switching valves, extra pumps, and trapping columns. Notably, only a few applications employ off-line 2D-LC, indicating that on-line 2D-LC is more suited and hence more desirable for pharmaceutical analysis. This is possible because, despite its benefits, off-line 2D-LC is tedious and cannot be automated [17, 18, 20, 22].

With stop and-go method, elution from the first-dimension column is prevented while a fraction is transferred to and processed on the second-dimension column, and then continued in the first-dimension. This reduces the second-dimension time limitations but increases peak parking periods, reducing first-dimensional separation efficiency. This method has been applied effectively, most importantly in multi-dimensional protein identification technology (MPIT). The benefit of a stop-and-go strategy is that the second dimension may be much longer than an online approach [19, 22, 23, 24].

In off-line approach, eluting portions are collected at regular intervals for further separation on the second dimension. Since there is no direct connection, samples may be desalted and/or recrystallized after the initial separation, making it possible to combine chromatography that is not directly compatible. There are several ways to alter the portions (dilution or concentration or dissolution in various solvents), chemically modify them, and analyze them again in order to improve their peak capacity as the second dimension’s analysis duration is unrestricted. As an additional benefit, a 2DLC separation may be performed using just one-liquid chromatography. However, this method is time-consuming and requires labor-intensive sample manipulation steps, making it is more susceptible to sample loss and contamination compared with other approaches [9, 25, 26].

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4. Fields of application of multi-dimensional liquid chromatography

Several multi-dimensional chromatography systems have been introduced in the last few years to improve the separation and perform an in-depth analysis of proteome and lipidomics, environmental chemicals, polymer and oligomer separation, metabolomics, and closely related medications (chiral drugs). The range of applications for MDLC is far too broad to be covered here. So, this chapter mainly focuses on pharmaceutical applications. It explains specific field of application with few practical examples. It also presents systematically gathered scientific information from a plethora of articles scattered over a wide a range of sources.

4.1 Pharmaceuticals

In the pharmaceutical industry, detection of all synthesis-related impurities and degradation products present with the active pharmaceutical ingredient are of extreme importance. High-performance liquid chromatography has been the technique of choice for many years to assess the chemical purity of drug substances and products that are widely used in the pharmaceutical industry, from research and development stages to quality control laboratories. The peak capacity and selectivity of this conventional liquid chromatography may not be sufficient to separate all substances. The implementation of MDLC is therefore highly beneficial in order to address co-elution issues and for the verification of chiral purity of the API. To further improve the probability of success, the chromatographic peaks can be analyzed on more than one orthogonal LC system. This can be performed by utilizing a second-dimensional screening module that comprises various column types, organic modifiers, and pH adjustments [11, 12, 22, 27].

4.1.1 Trace analysis

Peak co-elution is a significant problem in pharmaceutical analysis since impurities can co-elute with API or other components. One of the most applications of 2D-LC is directed toward the separation of peaks that co-elutes in conventional 1D-LC methods. This is of prime importance for peak purity assessment. This problem can be overcome using LC–LC with a heart cut of the fraction containing API and its co-eluted impurities [10, 13, 14].

4.1.2 Chiral analysis

Since many drugs are chiral, separation of them is gaining importance especially for those with two or more chiral center race mates. For separating enantiomers, heart-cutting (or multi heart cutting) liquid chromatography can be useful. The majority of research on direct chiral separations has concentrated on analytes in a basic sample matrix, and there has been relatively little research on the direct separation of medication enantiomers in biological materials using multi-dimensional chromatography. However, some publications are available on over a wide a range of sources and is presented in Table 3 [15, 16, 47].

CouplingAnalytesMatrixFirst dimensionSecond dimension2D-LC modeReferences
Trace analysis (determination of drug traces into biological matrices)
RPLC-RPLCTolterodine,
Amperozide & metabolites
Biological matricesAmide column Gradient elution
Ammonium formate pH 3.6 /ACN
F: 0.2 mL/min, T: 40°C
PFPP column Gradient elution Ammonium formate pH 3.6 /ACN
F: 0.2 mL/min
Heart cut[28]
RPLC-RPLCAPI & related isomer APIResearch compounds synthesizedEclipse XDB-C18 gradient elution
Water +0.1% FA/ACN F:1 mL/min, T: 25°C
XSelect CSH Phenyl-Hexyl gradient elution Water/ACN + 0.1% FAHeart-cut[18]
RPLC-RPLCAPI & metabolitesRats excretaXTerra TM RP18 Gradient elution Water 0.1% FA/ACN
F: 60 mL/min
T: 40°C
Discovery C18 Gradient elution Water +10 mM
ammonium acetate/ACN
− T: 40°C
Heart-cut (Off-line)[22]
RPLC-RPLCPhenprocoumon and metabolitesHuman plasmaGrom Sil ODS-3 CP C18 Gradient elution Ammonium acetate
buffer pH 3.9/MeOH
F: 200 μL/min
T: 40°C
Chira Grom 2 Isocratic elution Water/ACN + FAA pH 3.9,
F: 150 μl/min
T: 40°C
Heart cut[20]
RPLC-RPLCTerbutalineHuman plasmaNucleosil-phenyl isocratic elution ammonium acetate
pH 4.6
F: 0.2 mL/min
T: 40°C
b-Cyclodextrin
Isocratic elution
ammonium acetate
pH 6/MeOH
F: 0.7 mL/min
T: 40°C
Heart cut[15]
RPLC-RPLCAPI & impuritiesStandard mixturesZorbax Eclipse XDB C18 Gradient elution potassium phosphate
pH 6.5 in water/ACN
F: 1.2 mL/min
T: 40°C
Zorbax Eclipse XDB C18 Gradient elution
Water +0.05% FA/ACN
F: 1.2 mL/min
T: 40°C
Heart cut[21]
RPLC-RPLCDuloxetine &
impurities
Standard mixtureZorbax Eclipse Plus C18
Isocratic elution Phosphate
buffer/ACN/MeOH F: 1 mL/min
Zorbax Eclipse Plus C18
Isocratic elution
Water/ACN + 0.1% formic acid
F: As gradient
Heart cut[29]
RPLC-RPLCTaxanesStandard mixtures/extract
samples
Eclipse Plus C18 Gradient elution Water/MeOH
F: 0.06 mL/min
T: 30°C
Eclipse Plus Phenyl-Hexyl Gradient elution Water/ACN + FA
F: 4 mL/min
T: 40°C
Comprehensive[30]
IPC-RPLCPhenylpropanolamineHuman
plasma/urine
YMC ODS Isocratic elution phosphate buffer pH:3.5 + SBS/ACNF: F;1 mL/min—T: 40°CYMC ODS Isocratic elution phosphate buffer
pH 3.5/CAN F: 1 mL/min—T: 40°C
Heart cut[31]
NPLC–NPLCDexamethasoneBovine tissueSpherisorb phenyl Isocratic elution Water/acetic
acid/2-propanol/ hexane F: 1.5 mL/min
T: 30°C
Spherisorb CN Isocratic elution Water/acetic acid/2-propanol/ hexane F: 1.5 mL/min
T: 30°C
Heart cut[32]
RPLC-RPLC-RPLCMefenamic acidHuman serumYMC ODS Isocratic elution Phosphate buffer
pH:5/ACN F: 1 mL/min—T: 40°C
YMC ODS (1)—ODS 80 TM (2) Isocratic elution (1) Phosphate buffer pH 3.5/ACN (2) Phosphate buffer pH 6/ACN F: 1 mL/min—T: 40°CHeart cut[33]
RPLC-RPLC1,2,3,4-tetrahydro-1-naphthol (THN), hexobarbital (HXL)Pharmaceutical drug development samples.BEH C18, (150 mm × 2.1 mm × 1.7 μm), acetonitrile, 0.1% formic acid pH = 2
F: 0.15 mL/min
T: 25°C
C18 (150 mm × 4.6 mm × column packed with 3 μm particles)), 15 mM NH4TFA buffer acetonitrile, pH = 2 F: 0.25 and 1 mL/min T: 30°CHeart cut[34]
RPLC-RPLCAPI & impuritiesStandard mixtureXTerra RP18
column (4.6 × 150 mm, 3.5 m), mobile phase 10 mM potassium phosphate in water, pH 2.6 and ACN, F:1.0 mL/min, T: 30°C
C18 column (4.6 × 150 mm, 3.5 m) mobile phase 0.03% formic acid in water and CAN pH 4.0, F: 1.0 mL/min, T: 30°CHeart cut[21]
RPLC−RPLCVancomycinStandard mixture/Human plasmaRP ASTON C18
Isocratic elution Water/ACN + ammonium acetate pH:3.8
F: 1 mL/min
ACR C18 Isocratic elution
Water/ACN + ammonium
acetate pH: 5.2
F: 1.2 mL/min
Heart cut[35]
Chiral analysis
RPLC-RPLCKetorolac & ParacetamolHuman PlasmaDiscovery C18 Gradient elution Water +0.1% FA/ACN
F: 0.2 mL/min
ChiralPak AD-RH Isocratic elution
Water +0.1% FA/ACN
F: 0.15 mL/min
Heart cut[36]
IEX-RPLCPropafenoneHuman PlasmaPartisil SCX Gradient elution Water + Perchloric and phosphoric acids pH 2.4/ACN F: 0.7 mL/minChiralcel ODR Isocratic elution Water + Perchloric and phosphoric acids pH 2.4/ACN F: 1.3 mL/min—T: 15°CHeart cut[37]
HILIC-HILICSalbutamol, Salmeterol, AtenololUrine samplesKinetex HILIC Isocratic elution Acetate buffer pH: 6 /MeOH/ACN F: 0.4 mL/min
T: 25°C
Isocratic elution Acetate buffer pH: 4 /MeOH
F: 0.4 mL/min
T: 25°C
Heart cut[38]
NPLC-NPLCPimobendan & metabolitesHuman plasmaSpheri-5 silicaIsocratic elution A: n-hexane/EtOH +0.1%Et2NHF: F:1 mL/min—T: 35°CChiralcel ODIsocratic elution A: n-hexane/EtOH +0.1%Et2NH
F: 1 mL/min—T: 35°C
Heart cut[39]
Capillary extraction column-NPLCAntidepressant and Antiepileptic drugsUrine samplesGraphene Oxide Capillary Column, (100 mm × 2.1 mm × 2.7 μm dp), H2O/ACN (30%:70% 35%:65%), F: 0.20 mL/min, T: 40°CC8,(200-mm length and 508-μm
i.d.),H2O/ACN(30%:70%, 35%:65%)
, F: 0.05 mL/ min, T:150°C
Heart cut[40]
sRPLC-SFCChiral active pharmaceutical ingredient elated
Impurities
Complex pharmaceutical sampleAcquity BEH C18, Chiralpak IC column (50 mm × 2.1 mm 1.7 μm) MeOH/H2O 98/2 (v/v) F: 0.2 mL/min T: 40°C°CChiralpak IC column (150 mm × 4.6 mm; 3
μm), MeOH/H2O 98/2 (v/v) F:2.5 mL/min, T: 40°C
Multiple Heart-cutting[41]
RPLC-RPLCHydroxywarfarins and warfarinHuman plasmaRP Acquity UPLC BEH Phenyl column (2.1 mm × 150 mm 1.7 μm) particle column, F:300 μL/min, T:60°C.Chirobiotic V column [2.1 mm × 150 mm, 5 μm), room T: 21.6–22.4°CHeart cut[42]
RPLC-RPLCStereoisomers (from anti-HCV therapeutic)Resarch compounds synthesizedCortecs C18 Gradient elution
Water +0,1%
Phospohoric acid/ACN/MeOH F: 0,22 mL/min—T: 40°C
Teicoplanin Isocratic elution
Water +0,1% Phosphoric acid/ACN
Heart cut[43]
RPLC−RPLCLeucovorinDog plasmaResolvosil BSA-7 Isocratic elution Sodium phosphate in water pH 5.1 F: 1 mL/min T: 40°CLiChrocart RP-18 gradient elution Sodium phosphate in
water pH 5 /MeOH F: 1 mL/min T: ambiant
Heart cut[44]
RPLC−NPLCAPI & impuritiesResearch compounds synthesizedSymmetry Shield RP18 gradient elution Water +0.1% ortho-phosphoric acid/ACN F: 0.8 mL/min − T: 22°CChiralpak AD-H
Isocratic elution hexane/isopropanol
Heart cut[45]
RPLC−RPLCOmeprazoleHuman plasmaRestricted access media BSA octyl column Luna C8(100mm × 4.6 mm i.d., 10 μm) Water and ACN (30:70,v/v) F: 1 mL/minAmylos tris(3,5 dimethylcarbamate) (150 mm × 4.6 mm i.d., 7 μm) Water and ACN (60:40,v/v) F: 0.5 mL/minDirect Heart cut[46]

Table 3.

Multi-dimensional LC analysis in pharmaceutical products (trace and chiral analysis).

RP: reversed phase, F: flow rate, T: temperature, FA: formic acid, BSA: bovine serum albumin, ACN: acetonitrile.

BEH: ethylene bridge hybride, EtOH: ethanol, MeOH: Methano.

4.1.3 Separations of biopharmaceuticals

Biopharmaceuticals are therapeutic proteins produced in vivo through recombinant DNA technology and are generally used for the treatment of severe diseases, such as cancer, autoimmune disorders, and cardiovascular diseases. Several kinds of therapeutics fall within the category of protein biopharmaceuticals (hormones, growth factors, blood factors, vaccines, anticoagulants, cytokines, and others), but monoclonal antibodies represent the largest percentage of these drugs (mAbs) followed closely by mAb-related products, such as antibody-drug conjugates (ADCs). This biopharmaceutical examines latest research on using biologics to develop new drugs, vaccines, and gene therapies in the quest to realize the promise of personalized medicine [28, 48, 49].

Several researches have arisen in recent years, in response to this useful therapeutic area, using heart cutting, multiple heart cuttings, and comprehensive 2D-LC. A tryptic digest of trastuzumab was analyzed by three different 2D-LC combinations, including CEX × RPLC, RPLC × RPLC, and HILIC × RPLC, with both UV (DAD) and MS detections. The orthogonal information obtained by the application of the different LC × LC approaches allowed for assessing both the identity and purity of the sample. Similarly, the therapeutic monoclonal antibody, herceptin is characterized by different chromatographic approaches (RPLC, HIC, SEC, CEX, and HILIC). Similarly, HIC × RPLC–HRMS was performed to obtain and profile the drug-to-antibody ratio (DAR) of brentuximab vedotin in the first dimension (HIC) with an inline desalting step performed in the second dimension (RPLC) prior to the coupling with MS that allowed accurate identification of positional isomers. Another method was developed for streamlined characterization of an antibody-drug conjugate by 2D and 4D-LC/ MS. A 4D-LC/MS method (SEC-reduction-digestion-RPHPLC) was also developed to determine the levels of potential critical quality attributes (pCQAs) including aggregation, average DAR, oxidation, and deamidation in 2 h. With multi-dimensional liquid chromatography, different classes of multi-product mAbs (cetuximab, panitumumab, rituximab) separation are also feasible in both elution modes with generic salt and pH gradient CEX separation [29, 30, 31, 32, 33, 50, 51].

From the chiral analysis review, one study describes a 2D LC–MS approach that allows the simultaneous analysis of paracetamol and the two ketorolac enantiomers. Ketorolac is a non-steroidal anti-inflammatory drug (NSAID), which has a strong analgesic activity. It possesses a chiral center and is marketed as a racemic mixture of (+) R and (−) S enantiomers. The efficacy of combining paracetamol and ketorolac on numerous experimental pain models was evaluated in randomized placebo-controlled clinical trials in healthy human volunteers. As a result, an assay was needed to confirm the presence of these medicines in human plasma in order to characterize their pharmacokinetic profiles. The method consists of a gradient RPLC method on a C18 column coupled with a stereoselective, isocratic, RPLC method on polysaccharide chiral column, and the result explained in Figure 1 [36].

Figure 1.

Plasma concentration-time profile of (A) paracetamol (B) S and R-ketorolac, and (C) simultaneous analysis of both drugs after IV administration based on achiral-chiral 2D-LC.

Another instructive example is the combination of achiral and chiral separations in a single mLC–LC separation of warfarin and hydroxywarfarin isomers, where the first dimension resolves the majority of the analytes and the second dimension completes the job. The first dimension has a chiral stationary phase, whereas the second dimension has an achiral separation. This indicates that neither achiral nor chiral separation is sufficient to resolve the mixture. When the same separation modes are used in a multiple heart-cutting 2D-LC separation, the achiral reversed-phase separation used in the second dimension is highly complementary to the chiral separation used in the first dimension. These provide enough selectivity to quickly resolve those components of the sample that remains unresolved after passing through the 1D column [29, 34].

Another popular pharmaceutical application is comprehensive LC in stress-testing studies for different pharmaceutical products. The API’s and intrinsic stability of other drug products should be assessed, and the degradation mechanisms should be disclosed. Stress testing, also called forced degradation study, helps to establish the intrinsic stability of the API. Stress testing includes a number of experiments, such as the effect of temperature, humidity, oxidation, photolysis, or hydrolysis at different pH values, as outlined by the World Health Organization [33, 35, 36, 52].

The 1D-LC analysis of a strongly stressed (temperature, UV irradiation, and organic solvent) omeprazole tablet demonstrates this notion. One has the impression that in this study, the omeprazole peak is corresponding to only one product, the API, but recording a mass spectrum revealed that many solutes are co-eluting in the peak. Increasing resolution by utilizing longer columns or smaller particles is not a viable option because there is not enough peak capacity to resolve the solutes in the primary peak (zero resolution). Although choosing another stationary phase is an option, the risk of other peaks now overlapping is realistic. By far, the best solution is to combine different selectivities in an LC × LC combination even if the two mechanisms are comparable, for example, RPLC × RPLC but using different stationary and mobile phases [13, 35, 36].

Different mobile phase compositions with different selectivities were used in the first- and second-dimension columns. The study revealed more than 50 spots, with the omeprazole peak resolved in the second dimension into four distinct products, and its mass spectrum is also recorded. The conditions developed in this 2D-LC approach can be considered more or less generic for detection of impurities in APIs, at least when eluting under reversed phase LC conditions. In this robust and repeatable method, good finds were also achieved for analyses of metoclopramide, acetaminophen, diclofenac, ibuprofen, and lidocaine [32, 33, 34, 53, 54, 55, 56].

4.2 Traditional medicines (TMs)

Regardless of the fact multi-dimensional chromatography is applicable to many traditional medicines, its application in traditional Chinese medicine is quite well researched. Traditional Chinese medicines are garnering attention in modern pharmaceutical institutes as a valuable resource for medication development. TCMs are extremely complex mixes having hundreds, if not thousands of components of various structures and concentrations, with only a few compounds responsible for specific pharmacological activity and/or toxicity. As a result, the use of latest analytical techniques is critical for the elucidation of the composition and quality control of TCMs [42, 43, 57, 58].

For the analysis of TCMs, a number of two-dimensional LCs have been designed with different column. Mostly, commercial columns with various separation techniques, such as strong cation-exchange chromatography or reverse phase liquid chromatography, have been coupled. Such coupled separation mode may reveal much more information on components due to a specific interaction with these bio-macromolecules of traditional medicines. In most of the cases, the two columns have been combined off-line, which makes the process simpler. However, a few are truly comprehensive (LC × LC). Ligusticum chuanxiong, Angelica sinensis, Swertia franchetiana, and Lonicera caprifolium are some of the Chinese traditional medicines that are separated and identified by multi-dimensional liquid chromatography. Analysis of a traditional Chinese medicine using a two-dimensional cyano octadecyl silyl system with an eight port valves with two sample loops and UV APCI MS detection also gave over 52 components [40, 41, 42, 57, 58].

4.3 Lipidomics

The wide and complex lipid composition in biological samples requires MDLC methodologies to sufficiently separate the lipids prior to MS or other detector characterization. This lipid separation usually demonstrated with normal phase, reversed phase, and silver ion chromatography. The application of MDLC in complex field of lipidomics is becoming of increasing significance. One typical example of these lipidomics application is analysis of egg yolk by 2D high-performance liquid chromatography-mass spectrometry for phosphatidylcholine. Phosphatidylcholine is the main phospholipid present in egg yolk. For characterization of the fatty acids composition in phosphatidylcholines molecules in egg yolk, an off-line LC × LC-ESI-MS/MS (preparative C18 column) method using a triple quadrupole mass spectrometry was used. The study identified phosphatidylcholines, which contains unsaturated fatty acids from both omega-3 and 6 groups. Phosphatidyl ethanolamine, lysophosphatidylglycerol, sphingomyelin, lysophosphatidylethanolamine, lysophosphatidylcholine, and glycerophosphorylcholine were also among the 13 phospholipid fractions evaluated by this method [53, 54, 59, 60, 61, 62].

4.4 Proteomics

MDLC has been used very successfully in proteomics for about two decades. These commonly comprehensive type separations, variant unique to peptide analysis, are known as multi-dimensional protein identification technology (MudPIT). Mass spectrometry has been widely used with as the most common detector. MDLC coupled with mass spectrometry is becoming increasingly important in proteome research owing to its high speed, high resolution, and high sensitivity. Recent proteomics technologies offer excellent separation and enormous data-gathering capabilities in the discovery of peptides and proteins, particularly disease-specific biomarkers in serum, plasma, urine, tissue, and other biological samples. The performance of multi-dimensional chromatography separation techniques compatible with MS, which are commonly used in proteomics applications, is summarized in the Table 4 [45, 46, 53].

3D-LC setupSample (protein amount)Identified proteinsIdentified unique peptidesMS time (hr)YearReference
SCX-HILIC-RPHeLa cells (500 μg)342411,9801262013[63]
SEC-HILIC-RPParacoccus denitrificans (8 mg)2627662015[64]
RP-RP-RPJurkat cells (720 μg)14,230251,1661892016[65]
SAX-RP-RPHEK 293 T cells (30 μg)822274,43220.42017[66]
SCX & SAX-RP RPHuman serum (−)862122018[62]

Table 4.

Performance of various off-line 3D-LC systems in protein analysis.

SAX: Strong Anion Exchange, SCX: Strong Cation Exchange HILIC: Hydrophilic Interaction.

Liquid Chromatography, HEK 293 T cells: Human embryonic kidney 293 cells.

The first applications of multi-dimensional liquid chromatography for environmental research were performed at the end of the twentieth century. This application was known as column switching mode, which is widely employed for the separation and analysis of pesticides. Pol and coworkers published the first studies on the use of a comprehensive two-dimensional liquid chromatography system, termed LC–LC, for the analysis of environmental samples in 2006. This analysis showed acidic compounds present in atmospheric aerosols were separated using a multi-dimensional chromatographic system for the first time, proving the capabilities of LC–LC systems to separate intricate mixtures that would be too difficult for 1D-LC [67].

One of the environmental contaminants is endocrine-disrupting compounds (EDCs) containing chemicals and hormones having endocrine-disrupting activity. This compound becomes critical emerging contaminants due to their presence in environmental waters and worries about probable detrimental adverse effects to wild life and humans. A study done in Czech Republic was used multi heart-cutting two-dimensional liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometry method for the determination of endocrine-disrupting compounds in water. Twenty real samples collected from the Loucka and the Svratka rivers were analyzed, and compounds were found in all Svratka samples (9.7–11.2 ng l−1 for -estradiol, 7.6–9.3 ng l−1 for estrone, and 24.6–38.7 ng l−1 for bisphenol A) [64, 65, 68].

Another novel multi-dimensional separation system based on online comprehensive two-dimensional liquid chromatography and hybrid high-resolution mass spectrometry has been developed for the qualitative screening analysis and characterization of wastewater sample. The core of the system is a consistently miniaturized two-dimensional liquid chromatography that makes the rapid second dimension compatible with mass spectrometry without the need for any flow split. Elevated temperature, ultrahigh pressure, and a superficially porous sub-3-μm stationary phase provide a fast second-dimensional separation and a sufficient sampling frequency without a first-dimensional flow stop. To seek data for a suspected target screening of a wastewater sample, 99 substances were added in the reference mix [62, 66].

Another novel method was developed utilizing LC × LC-ESI-TOF coupled MS for the determination of organic acids in atmospheric aerosols. They analyzed methanolic extracts of filters containing atmospheric aerosols and expected to find mainly polar organic compounds such as aliphatic, aromatic, and substituted carboxylic acids. Because of the combined knowledge of the elution pattern and the sensitive and accurate mass spectrum data, this innovative perspective liquid chromatography (LC-LC-TOF coupled MS) is proven to be an appropriate method for screening undiscovered acidic compounds [67, 69, 70].

4.5 Recent advances and future perspectives

Over the last decades, multi-dimensional liquid chromatography techniques have been extensively exploited with the latest significant improvements in terms of instrumental setup and availability of novel stationary phases. Nowadays, robust and full-featured instrumentations are available from most LC manufactures. Miniaturization and downscaling, switching valves as well as suitable software offer the possibility to adapt two-dimensional procedures and instrumentations will certainly continue in the future, and consequently, a significant rise of 2D LC systems is expected in several research fields [71].

Another new development in this area is Nano 2D-HPLC. 2D online nano-LC/MS was developed that substituted the inserted salt step gradient with an optimized semi-continuous pumped salt gradient, and also, 8-Isoprostaglandin F2α was measured from human urine. A microchip-based nano-HPLC was also used. So it will be the most applicable technique especially in the chiral resolution in the future [72, 73, 74, 75].

Currently, a multi-dimensional liquid chromatography also gives different opportunity to those challenging areas for the analysis. Recently, major developments are seen and attracted significant interest toward complex in the analysis of small, more complex molecules and biological products. For example, biopharmaceuticals such as monoclonal antibody (mAbs), interferons/cytokines, and vaccines are recently analyzed by the advanced instrumental technology of multi-dimensional liquid chromatography [76].

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5. Conclusion

Multi-dimensional liquid chromatography where a complex mixture is separated by facing different dimension is useful in several fields, especially in-depth analysis of proteome and lipidomics, environmental chemicals, food and pharmaceuticals industries. It boosts separation power, peak resolution, and reproducibility while increasing system complexity. The primary reason for multi-dimensional separations is that they provide a more effective and efficient method of generating high-peak capacities, and hence allowing for more comprehensive resolution of complicated mixtures. Separation and characterization of complicated mixtures are critical in a wide variety of fields that demand considerable separation power. Multi-dimensional separations envisaged that better understanding and application of multi-dimensional separations would open up opportunities for meaningful analyses of extremely complex samples and allowing the regular analysis of thousands of constituents from a single sample in a single run.

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Written By

Esayas Tesfaye, Tadele Eticha, Ariaya Hymete and Ayenew Ashenef

Submitted: 12 December 2021 Reviewed: 01 April 2022 Published: 06 July 2022