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

*Esayas Tesfaye, Tadele Eticha, Ariaya Hymete and Ayenew Ashenef*

#### **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 singledimensional 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 multidimensional 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 [2–5].

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 [5–8].

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. Onedimensional 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].

#### **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 *Perspective Chapter: Multi-Dimensional Liquid Chromatography - Principles and Applications DOI: http://dx.doi.org/10.5772/intechopen.104767*

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 seconddimensional separation system is a major factor in determining the total analysis time of comprehensive two-dimensional separation systems and its resolution [15–17].

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].


#### **Table 1.**

*Multi-dimensional liquid chromatography combination modes.*

#### **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/reversedphase 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** [16–18].

#### **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].

#### *Perspective Chapter: Multi-Dimensional Liquid Chromatography - Principles and Applications DOI: http://dx.doi.org/10.5772/intechopen.104767*

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 multidimensional liquid chromatography have its own advantages and disadvantages. One has its own superior field of application over the other [20–22].

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


#### **Table 2.**

*Comparison of key attributes of the major implementations of MDLC.*

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, 22–24].

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].

#### **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].

*Perspective Chapter: Multi-Dimensional Liquid Chromatography - Principles and Applications DOI: http://dx.doi.org/10.5772/intechopen.104767*

#### *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].

#### *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-toantibody 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 multidimensional 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 [29–33, 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 placebocontrolled 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


#### *Analytical Liquid Chromatography - New Perspectives*


*Perspective Chapter: Multi-Dimensional Liquid Chromatography - Principles and Applications DOI: http://dx.doi.org/10.5772/intechopen.104767*


*Analytical Liquid Chromatography - New Perspectives*

