Principles and New Advances

## **Chapter 1**

## Principles and Applications of Ultra-High-Performance Liquid Chromatography

*Feruza Ahmed,Tadele Eticha, Ariaya Hymete and Ayenew Ashenef*

## **Abstract**

The science of separation had advanced significantly with the development of ultra-high-performance liquid chromatography (UHPLC), a brand-new type of liquid chromatography. The need for the evolution of HPLC into UHPLC has been driven by the continuously evolving of packing material modifications that affect the separation of mixtures. The separation process of analytes is completed in a substantially decreased amount of time due to the lower particle sizes, which increases surface area of interaction allowing reduction of column length to one-third; thus, shorter columns are employed in UHPLC, which consequently causes the flow rate to be three times higher and subsequently reducing analysis time. Although UHPLC shares the same fundamental idea and instrument layout as HPLC, it differs from HPLC in that it produces narrow peaks and has high spectral quality, allowing for simple compound identification in a variety of analytical applications such as impurity profiling, product formulation, and improved analytical technique and method development. However, high back pressure in UHPLC might lead to decreased column life, and the instrument's higher price compared to HPLC are the disadvantages.

**Keywords:** high-performance liquid chromatography (HPLC), particle size, ultra-high-performance liquid chromatography (UHPLC), principles of chromatography, applications of chromatography

## **1. Introduction**

Chromatography is a separation method that distributes a mixture's components between a stationary phase and a mobile phase via several methods, including adsorption, partition, ion exchange, and others [1]. The most popular method to identify, measure, and separate the components in a mixture is liquid chromatography (LC). Later, LC developed into high-performance liquid chromatography (HPLC), which pushes solvents through a column under high pressure [2]. It is an effective LC method for separating mixtures. Additionally, it is utilized to identify and measure pharmaceuticals in biological fluids, final dosage forms, and during the drug research and discovery process. Instrumental developments have been made and are still being made to improve resolution and other separation-related properties such as speed and sensitivity [3].

The development of UHPLC is a result of the growing need for quick and ultraquick separation techniques that are more effective and have superior resolution [4]. UHPLC has ushered in a substantial shift by giving analysts new ways to get quick analytical separation techniques without compromising the high-quality outcomes previously attained by HPLC [5].

UHPLC has an astonishingly short analysis time and uses a very small amount of solvent as the mobile phase. Additionally, it significantly increases separation effectiveness and analyte mixture resolution. UHPLC uses column packing particle size of less than 2 microns as its key differentiator from conventional HPLC systems, which use particles between 2.5 and 10 microns in size. Because the smaller particles (2 microns) require a greater pressure to work with, UHPLC systems must be able to perform over 6000 psi, which is frequently the upper limit of conventional HPLCs [5, 6].

## **2. Principles of ultra-high-performance liquid chromatography**

The first UHPLC systems appeared in 2004 [7]. The fundamental idea behind this modification of HPLC is that efficiency is gained as column packing particle size lowers, a particle size reduction of less than 2 μm results in an improvement in efficiency that does not drop at higher linear velocities or flow rates [3, 5].

By boosting chromatographic resolution with the greatest number of resolvable peaks, UHPLC enhances the separation systems of LC. By using small amount of column packing materials and reduction of particle size, analysis becomes faster and more sensitive. Additionally, UHPLC has enhanced instrument designs [8]. It had a shorter analysis time of about 1.5 minutes and it reduces the mobile phase volume usage by at least 80% when compared to HPLC. In general, the development of UHPLC has brought a significant advantage for analysts by providing quick and accurate analytical separation findings [5].

## **3. Distinct instrumental designs of ultra-high-performance liquid chromatography**

The common components of UHPLC system are solvent delivery systems (Pumps), sample injection, columns, column managers, detectors [3].

#### **3.1 Solvent delivery system**

UHPLC systems regularly operate at 8000–15000 psi. The delivery system must also counterbalance for various solvents used in isocratic, linear and nonlinear gradient elution modes, and also for solvent compressibility for a wide range of pressures. The two major classifications of solvent delivery systems are constant pressure pump and constant volume pump [2, 9, 10]. Constant pressure is used for column packing while constant volume pump is mostly used in all common UHPLC applications. HPLC has a pump pressure of 40 Mpa, whereas the UHPLC has a pump pressure of 100 Mpa [7].

## **3.2 UHPLC columns**

UHPLC columns are short in length and have a 150 2.1 mm length and diameter dimension respectively with a smaller diameter that ranges from 1 to 2.2 mm [10, 11]. Capillary columns are particularly suitable for UHPLC systems due to lower heat generation and better heat tolerance capability. These columns can operate with pressures higher than 80,000 psi [2]. Charged surface hybrid particle technology, ethylene bridged hybrid particle technology [1, 5, 6], high strength silica particle technology, and peptide separation technology are most commonly used in the construction of columns used in UHPLC [7, 10, 12, 13].

## **3.3 Column manager**

The Column manager adjusts temperature from 10 to 90°C and switches automatically for up to 24 hours to four columns, each with a diameter of 2.1 mm internal diameter and length of 150 mm. It also has the ability to bypass channel for flow injections. Most UHPLC systems contain a binary solvent manager, sample manager with the column heater, detector, and non-compulsory sample organizer. The binary solvent manager employs two individual serial flow pumps to deliver a parallel binary gradient. There is a built-in solvent selection feature valves that allow one to pick among the availed up to four different solvents [14].

## **3.4 Sample injection**

The volume of the sample in UHPLC is usually 2–5 μl. Injection cycle time is 25 s without a wash and 60 s with a dual wash used to further decrease carry-overs. A variety of microtiter plate formats (deep well, mid height, or vials) can also be accommodated in a thermostatically controlled environment when analyte stability and conditions demand. Using the optional sample organizer, the sample manager can perform injection from up to 22 microtiter plates [3]. There is also a direct injection application for biological samples [15].

## **3.5 The detector**

The most common detectors used in UHPLC analysis are UV/visible-based types. Detection of analytes is conventionally based on absorbance [9]. The UHPLC detector ought to have long path length, low volume detection cell, a highest likely sensitive detection, and reliable quantification of the narrow peaks. System volumes should also be lessened to uphold the speed, resolution, and sensitivity of the analysis [10]. Depending on the type of detector, the sensitivity of UHPLC can be increased by 2–3 times more than that of HPLC [2].

## **4. Applications of UHPLC**

UHPLC is playing a substantial role in the advancements of liquid chromatography. This is highly attributed to its ability in providing efficient and fast analysis. It can also be hyphenated with different instruments that make application in immense territories like that of pharmaceutical, toxicological, and food industry. It is helping to determine the nutritional value of certain types of foods. It had wide applications in

different agricultural sectors and in clinical analysis, where it is vital to increase throughput with reduced analysis costs [16].

#### **4.1 Analysis of herbal medicines**

Herbal medicines are not pure products with a single active ingredient. Thus, conventional methods for screening and identifying the active ingredients in natural products are inefficient. Traditional natural product discovery, using conventional methods, does not give enough evidence about mode of action until late stage in the discovery course. This causes finding compounds with exceptional biological properties (single compound/API drug discovery) a difficult task [17].

Although herbal medicines are gaining increasing attention, clinical usage studies that evaluate its safety and efficacy based on *in vivo* pharmacokinetic data of its main ingredients are limited [18].

Chromatographic fingerprinting of herbal components by UHPLC–MS has become a powerful and widely used technique today. This is because it could systematically profile the composition of herbal medicine samples. It also provides high-quality separations and detection capabilities for active compounds in highly complex samples derived from natural origin herbal remedies [19–21]. The application of UHPLC-HRMS for quality control of traditional Chinese medicine (TCM) includes chemical characterization of TCM, determination of TCM components, chemical fingerprint analysis, identification of the authenticity of TCMs, identification of illegal additives in TCMs, exploring the Quality-Marker (Q-Marker), identification of metabolites, evaluation of the quality of TCMs from different habitats, elucidation of the mechanism of action of TCMs (10).

**Table 1** shows typical applications of UHPLC in the analysis of herbal medicines where different methods of UHPLC were employed.

The pulp, seeds, and peel of the seven tropical fruits were tested, and the antioxidant levels and capacities revealed notable variations. Fruits' peels and seeds contain more antioxidant potential and antioxidant chemicals than the pulp, which is deficient in these substances. Most of the samples' antioxidant component levels are reduced by oven and freeze drying process. The results of this study had shown how valuable sources of natural antioxidants in avocado-dried peel and passion fruit seed could be affected by the processing techniques. By using UHPLC-ESI-MS, organic acids (citric, malic, and tartaric) and phenolic compounds were also quantified [17].

By using UHPLC/QTOF MS together with automated identification by the MetabolynxTM instrument, a total of 33 peaks were tentatively identified *in vitro*, 24 of which were parent components, with nine metabolites being detected: This offered beneficial chemical data for additional pharmacological investigation [22].

The validated UHPLC-ESI-MS/MS method was used in the PK study of 14 ingredients after oral administration of Gumiganghwal-tang tablets, a TCM [18].

More importantly, the application of the UPHLC/MS-network pharmacology method will offer a more trustworthy and convincing tool to identify future prospective targets and biological processing mechanisms of Chinese medicine [19].

In an investigation, acetylcholinesterase inhibition assay *in vitro* were utilized to quickly screen and discover acetylcholinesterase inhibitors in the D. auriculatum using UHPLC CQ-TOF-MS and UHPLC-ESI-MS/MS. The technique could be utilized for the quick discovery of novel AChEI from natural compounds and was straightforward, sensitive, and selective. This study had provided scientific experimental basis for the traditional efficacy of the medicinal plant for neurological disease [20].


*Principles and Applications of Ultra-High-Performance Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110540*

> **Table 1.**

 *Application of UHPLC in natural and herbal medicines analysis.*

UHPLC has been employed to study a herb-drug interaction in animal experiments, that is, rats. It was used to evaluate the tissue distribution and excretion of *Polygonum capitatum* extract and Levofloxacin when co-administered on rats [21].

#### **4.2 Analysis of drugs in human plasma**

Detection of drugs in biological samples is a requisite to study the pharmacokinetics, toxicity, and bioequivalence of the medicine [5]. The most crucial part of determination of drugs in biological samples (plasma, serum, urine, saliva, etc.) is sample preparation. It is often a baseline for a reliable and fast approach in refining analytical efficiency. Blood sample contains numerous proteins and other possible interfering components that would affect the detection of analytes unless otherwise eliminated. Protein precipitation, solid-phase extraction (SPE), and liquid–liquid extraction (LLE) have been widely employed for sample preparation to recover sufficient analyte from the biological sample matrices [24].

For example, a method was developed and validated for drug adherence measurement of patients with hypertension. The antihypertensive drugs were analyzed from patients' blood using a simple and fast sample preparation protocol with protein precipitation followed by chromatographic separation using a gradient elution on a reversed phase column. Mass spectrometric detection was conducted by applying both positive and negative electrospray ionization (ESI+/ESI) and selected reaction monitoring mode (MS/MS). Only 50 μl of plasma sample was needed for the simultaneous quantification of all twelve compounds (Amlodipine besylate, hydrochlorothiazide, nifedipine, spironolactone, valsartan, canrenone, enalapril, losartan, losartan carboxylic acid, perindopril and perindoprilate, enalaprilat) within 6-min runtime. Enalapril-d5 was applied as internal standard for all compounds except hydrochlorothiazide in which case the internal standard was hydrochlorothiazide-13C,d2. The method showed a significant advantages of minimal sample volume, clean-up procedure, and a short runtime. The method is now available to monitor drug adherence of patients thus helping to manage resistant hypertension in a hospital setting [25].

Analysis of drugs in human plasma using UHPLC plays another significant role in the therapeutic drug monitoring (TDM) activities. The method is quite simple and rapid (separation of compounds achieved in only 6 min) using just a precipitation method for sample preparation and utilizing low amount of patient plasma (100 μL). Due to these characteristics, the UHPLC-based method allows processing of clinical samples and rendering of results within the same working day. This makes it advantageous and fit for TDM purposes [26].

UHPLC has also been employed in the forensic sector. It analyzes compounds of forensic interest in human plasma. A study was carried out on Diphenidol (DPN), a non-phenothiazinic antiemetic, and antivertigo drug following some cases of suicide and accidental poisoning in China. Even though the exact mechanism of death caused by DPN poisoning has not been fully explained yet it is thought that an excessive amount of DPN may promote successive H1 receptor antagonist and anticholinergic effects, causing depression in the central nervous system and vascular smooth muscle relaxation, resulting in sedation, lethargy, hypotension, convulsions, and respiratory failure. The study had been performed using UHPLC–MS–MS indicated that the postmortem concentration range of heart blood was 0.87 99 μg/mL. The method was successfully applied to the detection and quantification of DPN in 15 real forensic cases [27]. **Table 2** lists UHPLC's deployment in drug analysis from human plasma.

### *Principles and Applications of Ultra-High-Performance Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110540*

Traditional methods are typically time-consuming, non-quantitative, and complex, and the necessary sample size is usually big, making them challenging. To fulfill the requirements of quick clinical detection and forensic identification, it is crucial to design simple, sensitive, and accurate systems. UHPLC analytical technique was effectively utilized for quantitative analysis of diphenidol in tissues where blood samples are unavailable or of poor quality [27].

In comparison with previously published methods, the recently developed UHPLC approach is quick and inexpensive, and has superior selectivity and sensitivity for the simultaneous quantification and pharmacokinetics study of human medications in their pharmaceutical dose forms and in spiked human plasma [28–30].

#### **4.3 Pharmacokinetics and bioavailability study**

UHPLC method has been developed and validated for the quantitative measurement and determination of pharmacokinetics and bioavailability of various drugs. This had a significant task in assuring drugs' quality, safety, and efficacy. Besides, since most preclinical studies are done on animals, there has been an indication of potentially massive savings of the number of animals and the amount of compounds used when efficient UHPLC methods are employed [31].

UHPLC is also useful for the study of pharmacokinetics and bioavailability on herbal medicines. UHPLC–MS/MS method was developed for the determination of Uncaria alkaloids (a TCM of dry hooked branch of Rubiaceae) which has shown an antihypertensive, vasodilating, neuroprotective, antidepressant, antiarrhythmic, antiepileptic, and antitumor activities in the blood of mice. The relevant methodological process was verified using UHPLC analytical methods. The results showed that the established UHPLC–MS/MS method is accurate and fast. It takes only 5.5 min to analyze blood sample. It is highly sensitive, and effective for the detection and pharmacokinetic study of Uncaria alkaloids. The pharmacokinetic results showed that the six Uncaria alkaloids metabolized rapidly in mice with a half-life between 0.6 and 4.4 h. The bioavailability study also showed satisfactory oral absorption of each alkaloid [32]. Similar studies were performed on other different herbal medicines [33–35]. **Table 3** depicts typical examples of application of UHPLC in pharmacokinetics and bioavailability studies whereby chromatographic and mass settings are crucial for the method's high sensitivity and short retention period in addition to the extraction and sample preparation stages. With modern technology, such as ultraperformance liquid chromatography based on tandem mass spectrometry, it is now possible to detect drugs with great sensitivity and speed, especially for counterfeit medications with variations in the package or appearance. UHPLC–MS/MS analytical method development was used to investigate the rate of degradation of pharmaceuticals in microbial fuel cells (MFC). Additionally, by observing the voltage produced by the microorganisms and the rate of chemical oxygen demand (COD) elimination, the capability of MFC to treat urine spiked with medications was examined. The performance of MFC and medicinal additives was shown to be related, according to the findings [36].

A study done on Pimavanserin indicates that it passes the blood–brain barrier and approaches a Cmax of 21.96.66 ng/g in 2.0 hours, according to pharmacokinetics and brain uptake experiments using the technique. Additionally, it was discovered that the ratio of brain to plasma for pimavanserin (Kbrain/plasma) is 0.160.05 and that it is quickly removed. The developed method using UHPLC was linear (R2 > 0.99 over the range of 0.1–300 ng/mL in plasma and 0.25–300 ng/g) in the brain homogenate [39].


**Table 2.** *ApplicationofUHPLCinanalysisofdrugsinhumanplasma.*

**Table 3** describes the utilization of UHPLC for the study of pharmacokinetics of wide array of human medicines where the method was accurate, sensitive, and with a short analysis time.

### **4.4 Identification of metabolites (metabolomics)**

Metabolomics a field of studies dealing with identification of many different metabolites that give virtual devices a far-reaching opportunity of utilizations in the fields of pharmacology, toxicology, enzyme discovery, and systems biology [43]. It intends to identify and quantify the full complement of low-molecular-weight, soluble metabolites in actively metabolizing tissues [44].

It also has an extensive contribution in improving ones understanding of disease mechanisms and drug effects. It improves our ability to predict individual variation in drug response phenotypes. Substantial attention has been developed in the application of metabolomics to describe different pathological states of human diseases such as cancer, diabetes, autoimmune, and coronary diseases, among others [43]. Recent technological advances allow characterizing plenty of metabolites from a small quantity of a biological samples. Numerous experiments conducted on cells and tissue cultures played a significant role in improving our biomedical understanding. This at the same time provides a foundation to interpret the concerns related to metabolic processes of the test subject [45]. UHPLC is considered suitable for metabolite profiling and metabolomics among the various LC platforms, especially for large-scale untargeted metabolic profiling due to its sensitivity, selectivity, and enhanced reproducibility [44]. The sensitivity and selectivity of UHPLC at low detection levels produces a precise and reliable data that can be used for a variety of reasons including pharmacokinetics, toxicity, and bioequivalence studies of different metabolites [46].

A metabolism study is considered to be mandatory in a drug development process. It has a vital role in the development of a new chemical entity (NCE). It aids in identifying the active metabolites in so doing, monitoring the possibility of reactive metabolite formations, augmenting pharmacokinetic and pharmacodynamics properties, comparing preclinical with clinical metabolism profile, understanding clearance, and predicting drug–drug or food-drug interactions at early stages of drug discovery [47]. By the time a NCE reaches the development stage, its metabolites' characterization becomes a critical process. The feeble spots of metabolites of drug candidate molecules are recognized and protected by changing the compound structure early [5]. Various analytical techniques have been coupled with UHPLC for the study of metabolomics. From the possible coupled techniques with UHPLC, nuclear magnetic resonance, the most uniform detection technique, has a lower sensitivity compared to mass spectrometry (MS)—and thus, the detection ability of lowabundance metabolites is restricted. Therefore, high-resolution mass spectrometry (HR-MS) and tandem mass spectrometry (MS/MS) are widely used in metabolomics. HR-MS, such as quadrupole time-of-flight mass spectrometry (QTOFMS), provides accurate mass and specific fragment patterns of MS/MS, which can improve the speed and the efficiency of metabolite identification [48]. Higher sensitivity, greater resolution, and faster separation are all benefits of using UHPLC coupled with quadrupole time of flight tandem mass spectrometry (UHPLC-QTOF/MS) for the evaluation of modified metabolites in intricate components. With the assistance of UHPLC-QTOF-MS/MS, a total of eight metabolites of a newly discovered piperazinebased anticancer molecule (IMID-2) were found and described in various matrices



*Application of UHPLC in pharmacokinetics and bioavailability studies.*

### *Principles and Applications of Ultra-High-Performance Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110540*

including in rat liver microsomes (RLM), human liver microsomes (HLM) and rat S9 fraction (RS9), rat plasma, urine, and feces [47, 49]. Untargeted metabolomics in diseased plants may offer a fresh viewpoint and advance our comprehension of plant defense mechanisms. A relevant method for comparing plant metabolite changes is metabolomics. UHPLC paired with HR-MS is the most extensively used metabolomics technology due to its great sensitivity [48].

**Table 4** depicts examples in the study of metabolomics using UHPLC. One study investigated and identified the metabolic characteristics of serum samples from children with urolithiasis and normal controls through UHPLC–MS-based metabolomics study approach. Forty differential metabolites were identified, mainly involved in retinol metabolism, steroid hormone biosynthesis, and porphyrin and chlorophyll metabolism. These results indicated that the metabolic phenotype of serum in patients with kidney stones was significantly different from that found in normal controls. The study provided a new understanding into the potential pathogenesis of urolithiasis, which may help to develop novel therapeutic strategies and preventive interventions [53]. Metabolomics can also be used to identify and quantify metabolites from plants. UHPLC–MS method was developed for chemotaxonomic study of seed accessions belonging to 16 different species of *Vicia* (vetch species family Fabaceae-Faboideae). These seeds are produced and consumed worldwide for their nutritional value. Both domesticated and wild taxa were analyzed by chemometrics-based UHPLC–MS method. A total of 89 metabolites were observed in the examined *Vicia* accessions. Seventy-eight out of the 89 detected metabolites were annotated. The study shows UHPLC–MS metabolomics method ability to discern the diversity of metabolites at the intrageneric level among *Vicia* species [54].

The discoveries of metabolic pathways and metabolites could provide a certain theoretical basis for drug discovery and pharmacological mechanism illucidation research [50].

### **4.5 Detection of impurities**

Identification of impurities in raw materials and the final products is one of the most vital stages of a drug development process [4, 55]. Regulatory authorities give significant attention to impurity profiling. Starting materials, intermediates, precursors, etc., are the most common impurities found in every API unless proper care is taken in every step involved throughout the multi-step synthesis. Sometimes, impurities of intermediates and precursors generate structurally related by-products during synthesis [56].

HPLC with sufficient resolution has been providing an excellent detection and determination of the lowest level of impurities with highly reproducible results. However, due to the presence of excipients, there is prolonged HPLC analysis time so it becomes necessary to perform several analytical runs to get the required data time. Hence, the UHPLC/MS technique is operational at alternate low and high collision energies. The fast change of collision energy produces both precursor and product ions of all analytes present in the sample, which allows rapid identification and profiling of impurities [5]. **Table 5** shows several methods for determining, quantification, and characterization of pharmaceutical, impurities using UHPLC have been devised that are easy to use, quick, appropriate, precise, and accurate. In terms of specificity, system suitability, linearity, limit of detection and quantitation, accuracy, precision, robustness, and solution stability, the suggested technique was fully


 *in* 

*identification*

 *of metabolites.*


*Principles and Applications of Ultra-High-Performance Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110540*

> **Table 5.**

*(90:10v/v). π*

*potassium phosphate monobasic buffer and acetonitrile (10 mM, adjusted to pH 4.0 with 1%* 

*orthophosphoric*

 *acid.*

> *Application of UHPLC in detection of impurities.*

validated. The validation results also indicated positive data for each of the examined parameters [56–63].

#### **4.6 Rapid analysis of dosage formulations**

Since disease-causing microorganisms are in more mutation than ever and humans' lifestyle is causing more illness than before, the pharmaceutical industry is under intense pressure to increase productivity and bring new drugs onto the market in a short period. UHPLC system provides accurate and reproducible results in rapid isocratic and gradient methods for drug molecules analysis in dosage forms [34]. This in turn helps in improving pharmaceutical manufacturing efficiency. **Table 6** shows examples of UHPLC application in the analysis of dose formulation.

#### **4.7 Food safety**

Food products'safety and quality are a concern for consumers and governments. Analytical information, including surveillance data for both recognized and newly identified contaminants, is also indispensable. However, information about food contamination incidence is still limited [65].

Food additives and mycotoxins are among the major toxic treats in different foods consumed worldwide [64]. Food stuffs are vulnerable to infection and contamination in the field or during storage. Mycotoxins, for instance aflatoxins (Afs), Afs B1 is the most common aflatoxin, and is the most toxic and carcinogenic and ochratoxin A (OTA) along with OTA C is known to cause hepatotoxicity, immunotoxicity, neurotoxicity, teratogenicity, and carcinogenicity. The challenge imposed on food safety due to mycotoxins is given currently a rising attention by the government, regulatory authority, and academia globally [66].

The safety of food components and contaminants can only be guaranteed when a good analysis approach is available. During the past few decades, chromatography has been recognized as one of the methods employed to identify and quantify food contaminants. For both qualitative and quantitative research, this novel procedure enables the isolation, purification, and detection of components from a mixture. UHPLC–MS has lately been utilized to estimate food pollutants and components in order to improve food safety [64].

Sensitivity of UHPLC has reached ppb and ppt levels; thus by these quality analytical results, a food analyst would be more confident in ensuring safe food consumption [65]. It is also ideal for analyzing low-level concentrations of food additives where high sample throughput is required without affecting the method's accuracy and sensitivity [67]. **Table 7** shows applying the rules in the context of complicated matrices, like coffee, which frequently necessitates costly and time-consuming strategies. Without clean-up, the UHPLC–MS/MS approach offered adequate sensitivity and resolution. The performance attributes of the approach include high LOQ, recovery, and precision [66]. Additionally, the UHPLC-ESI-MS/MS technology is utilized to discover other potential microbial metabolites present in samples and to confirm the identity of mycotoxins that have been detected [68]. UHPLC has also been employed to detect adulterated milk powder samples, with an acceptable linear correlation coefficient. It is used to assess food products and their diversity or assure their quality and authenticity [69, 70]. Quantification of essential nutrients from human milk was quantified by UHPLC to direct the diet for lactating women [72].

*Principles and Applications of Ultra-High-Performance Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110540*


**Table 6.**

*Applications of UHPLC in analysis of dosage formulations.*


**Table 7.** *Application*

 *of UHPLC in food safety* 

*determination.*

*Principles and Applications of Ultra-High-Performance Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110540*

### **4.8 Application in agricultural sector**

UHPLC has also been useful in the agricultural sector for the study of soil components, pesticide residue analysis, and crop analysis. Agricultural products contain not only plant materials but animal products too. Thus, safety and quality of such products need to be studied and quality ensured before consumption [74, 75]. In this direction, a rapid, sensitive, and specific ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC–MS) method was developed for the analysis of tetracycline antibiotics, including tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and their 4-epimers (4-epi TCs) in agricultural soil fertilized with swine manure. The limits of detection for the soil extraction method ranged from 0.6 to 2.5 μg kg<sup>1</sup> with recoveries of 23.3–159.2%. The method was applied on an agricultural field in an area with an intensive pig-fattening farm. Tetracyclines detected in the soil varied from 2.8 to 42.4 μg kg<sup>1</sup> . The results made evident that soil from swine farms can be rigorously contaminated with tetracycline antibiotics and their metabolites [74]. **Table 8** summarizes the effect of fungicide and pesticide treatment on the agricultural lands in which case they were considered and quantified by analyzing the harvested products using UHPLC. Besides seasonal variations on the harvested food, both occurrence and concentration of antioxidant abilities and flavonoids were investigated using UHPLC [75–77].

#### **4.9 Method development and validation**

UHPLC has been used broadly for method development and validation purpose. UHPLC plays a crucial role in fundamental laboratory function by increasing efficiency, reducing costs, and improving opportunities for business success. Using UHPLC, analysis times become as short as one minute, methods can be optimized in just one or two hours, thereby appreciably decreasing the time required to develop and validate new analytical method [79]. As an example, a study done for the simultaneous estimation of paracetamol and caffeine capsules dosage form indicated that a method was validated using various validation parameters such as accuracy, precision, linearity, and specificity. Moreover, the results show that the method could find practical application as a quality control tool for analyzing the drug in its capsule dosage forms in pharmaceutical industries offering the above-mentioned advantages [80].

## **5. Advancements of UHPLC**

UHPLC is thought to provide higher rates of efficiency, sensitivity, speed, and resolution of UHPLC devices make the system perfect for use with mass spectrometer. More laboratories are finding UHPLC–MS systems to be practicable due to the accessibility and low cost of a new generation of MS equipment [81]. The software used and created for UHPLC systems, which makes the instrument easy to manage, diagnose, and monitor, is primarily responsible for another breakthrough. This makes a significant contribution to the dynamic data processing and information management that convert the findings of the UHPLC system into useful knowledge. In addition, the recording technology found on the majority of UHPLC columns allows for the recording of column history. Another important development is the availability of sample organizers that multiply system capacity by more than 10 times. Additionally,



#### **Table 8.**

*Application of UHPLC in agricultural sector.*

## *Principles and Applications of Ultra-High-Performance Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110540*

some businesses offer a virtual technical support service, which enables them to give customers prompt, proactive help that meets their needs to the fullest extent [14].

Examples of organizers that boost system capacity more than ten times are technologically availed. Thus, UHPLC–MS systems are becoming more practical for more laboratories [82, 83]. Despite its expensive price, these developments will further elevate the UHPLC as a crucial analytical tool for sample studies that will be the analysts' first choice worldwide. Future research on engineering and material science topics might also be able to resolve this issue, making this analytical instrument more accessible to many laboratories.

## **6. Conclusion**

Liquid chromatography has been replaced by UHPLC in a novel approach of improvement. It is a sort of separation technique with nearly identical principles as HPLC. The UHPLC uses small packing particles (less than 2 μm in size), which directly influence the length of the column and, in turn, minimize solvent consumption and shorten analysis times. The quick analysis makes it possible to do several analytical tasks quickly. Additionally, UHPLC has been a popular option for analytical work in recent years due to its better sensitivity and resolution. It is used in many fields because of the excellent quality outcomes that are produced. In addition, UHPLC had a wide range of applications in the fields of bioavailability, pharmacokinetics, natural medicines, metabolomics, food safety, and agricultural sectors. The pharmaceutical industry is benefiting most in the drug discovery process. With the aid of UHPLC, researchers may now complete a range of investigations in a short duration of time while saving money on analysis.

## **Author details**

Feruza Ahmed<sup>1</sup> , Tadele Eticha<sup>1</sup> , Ariaya Hymete<sup>1</sup> and Ayenew Ashenef1,2\*

1 Department of Pharmaceutical Chemistry, School of Pharmacy, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia

2 Center for Innovative Drug Development and Therapeutic Trials for Africa (CDT-Africa), College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia

\*Address all correspondence to: ayenew.ashenef@aau.edu.et

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## **Chapter 2**

## Turbulent Flow Chromatography: A Unique Two-Dimensional Liquid Chromatography

*Francesca Di Gaudio, Annamaria Cucina and Sergio Indelicato*

## **Abstract**

Among 2D-LC techniques, a particular approach is commercialized by Thermo Fisher Scientific that may enable the direct introduction of biological samples into an online automated extraction system without any additional pre-treatment: the TurboFlow technology. It combines chemical and size exclusion capability of chromatography columns packed with porous particles in which a turbulent solvent flow is able to separate smaller molecules from larger ones (e.g. proteins). Once extracted, the small molecules can also be transferred to an analytical column for improving separation prior to detection. This is done through a unique plumbing and customized valve-switching arrangement that allows the focusing of molecules onto the second column. This enables a very efficient chromatographic separation. The use of the TurboFlow not only eliminates extensive sample preparation, thus reducing interoperator variability and matrix effects, but also increases the capacity for highthroughput analyses due to a unique multiplexing technology, in which multiple LC channels are connected to a single detector.

**Keywords:** 2D-LC, turbulent flow chromatography, TurboFlow, multiplexing, extraction, purification

## **1. Introduction**

High-performance liquid chromatography (HPLC) is a powerful technique for the separation of compounds. However, one-dimensional liquid chromatography (1D-LC) cannot easily handle complex matrices and complex mixtures of analytes. The matrix effect is relevant when dealing with biological matrices, such as urine, saliva, serum, and whole blood in liquid chromatography coupled with mass spectrometry (LC–MS). This effect is mainly due to co-elution of endogenous compounds, such as proteins, lipids, sugars, or salts [1, 2]. In fact, early approaches in LC–MS, tending to simplify sample preparation methods, such as "dilute and shoot" and quick chromatographic analysis time, soon revealed that by not removing the matrix components, these could interfere with the ionization process in an unpredictable and inconsistent way (e.g. ion suppression).

For these reasons, a thorough and robust sample preparation is crucial for quantitative analysis. Among others, protein precipitation (PP) methods are widely used. In these procedures, a small volume of sample is mixed with a certain volume of protein precipitation reagent. The interaction between proteins and this reagent determines the alteration of the protein conformation and the consequent precipitation. Once the precipitate is separated, the analytes of interest, remaining in solution, are ready for analysis. Although PP protocols are inexpensive and simple, they are time-consuming and unable to purify the samples enough. More recently, modified PP approaches, such as membrane-based PP filter plates, have been proposed to overcome these limitations [3, 4].

Liquid–liquid extraction (LLE) and solid-phase extraction (SPE) are alternative sample preparation methods. The first one consists in extracting the analytes from one liquid phase to another immiscible liquid phase. Once shaken or vortex-mixed, the phases are separated, and the molecules collected for analysis. LLE presents several limitations, such as the need for large sample volume, variable recovery, and difficulty on the extraction of compounds with varying lipophilicities. A more efficient upgrade of this technique is represented by salting-out assisted LLE (SALLE), in which appropriate salts are added to the solvent to enhance the extraction [5].

SPE technique allows extraction and enrichment of analytes of interest by loading a liquid sample onto a column/cartridge/plate packed with a sorbent material. While the analytes are retained, the interferents are either eliminated during the loading phase or washed away during the washing steps. The analytes are then eluted and collected for LC–MS analysis [6]. Even if SPE has many advantages, it has limited selectivity and sensitivity because matrix constituents can be adsorbed together with the analytes, determining matrix effects. Furthermore, SPE cartridges are usually single use. More recently, several approaches have been developed to improve SPE technique, such as dispersive solid-phase extraction, solid-phase micro-extraction, and stir bar sorptive extraction [7]. Furthermore, SPE has been automated in online solid phase extraction, coupled with HPLC [8]. This technique can thus be considered as an online two-dimensional liquid chromatography (2D-LC).

The online 2-D approaches are faster and more reproducible, but they require specific interfaces and more complex operative modes.

There are two main approaches to the online 2D-LC: heart-cutting and comprehensive [9]. The first one enables the re-injection of a definite number of multicomponent effluent fractions from a primary to a secondary column, while the second one determines the separation of the entire sample in both dimensions [10]. When this technique is employed, a combination of different chromatographic separation methods is usually utilized. Frequent combinations are size exclusion chromatography (SEC) with reverse phase liquid chromatography (RPLC), RPLC with hydrophilic interaction liquid chromatography (HILIC), or normal-phase liquid chromatography with RPLC [11].

Regardless of the operative mode, 2D-LC presents some disadvantages. In fact, the separation typically takes longer than in 1D-LC, and the detection sensitivity may decrease because of the dilution or loss of the sample during the two separations. In addition, considering that the 1D effluent is the injection solvent of the second dimension, incompatibility issues may be raised. Incompatibility could be determined by partial or complete immiscibility of the two mobile phases, difference in solvent strength, but also by the excessive difference in viscosity, that could result in peak deformation or splitting. Besides, an increased instrumentation and conceptual complexity must be considered. Active-modulation techniques, such as active solvent modulation (ASM) and stationary-phase assisted modulation (SPAM), could surmount these limitations. However, a constraint to the ASM approach is the time

*Turbulent Flow Chromatography: A Unique Two-Dimensional Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110427*

needed to displace the fractions of effluent from the sampling loop to the second column. The required extra-time can be significant for short 2D cycles [12]. SPAM separation methods of analytes with completely different chemical properties could lead to incomplete recovery and discrimination effects, due to a partial trapping of all analytes with the chosen dilution solvent. Moreover, the trapping columns of SPAM could reduce the robustness of the system [13].

Turbulent flow chromatography (TFC) overcomes the limitations of 2D-LC by removing potential interferences in a fast and efficient manner. In addition, the application of staggered, parallel methods ("multiplexing") maximizes the use of detector time, saving time and solvents. Furthermore, the extraction columns are re-usable for hundred injections [14].

In this chapter, theory and hardware aspects of the TFC, with particular reference to the TurboFlow system, will be discussed. Moreover, method development and applications will be brought up.

## **2. Theory of turbulent flow chromatography**

In the ideal chromatographic process, the analytes form narrow bands while moving along the column. The band or peak narrowness is a measure of the efficiency of the separation procedure that can be quantified by the number of theoretical plates, *N*. These latter are hypothetical zones or stages in which two phases, the stationary phase, and the liquid mobile phase in the case of HPLC, establish an equilibrium with each other. If the peak shape is Gaussian, the theoretical plate number *N* can be approximated according to Eq. (1).

$$N = 5.54 \ast \left(\frac{t(r)}{W(1/2)}\right)^2\tag{1}$$

where *t(r)* is the retention time of a compound, and *W(1/2)* is the peak width measured at half of the peak height. As described in the Eq. (2), the theoretical plate number *N* depends on the ratio between the column length and the height equivalent of a theoretical plate, *h*.

$$N = \frac{L}{h \ast d(p)}\tag{2}$$

where *L* is the column length, *N* is the number of theoretical plates, and *d(p) is* the particle diameter. The smallest value of *h* corresponds to the highest efficiency. Thus, *h* is a more effective way to compare efficiency of the chromatographic processes.

Column efficiency, linked to band broadening processes, in traditional HPLC was first described by van Deemter and colleagues [15]. Van Deemter described these mechanisms with the Eq. (3).

$$h = A + \frac{B}{v} + \text{C}v \tag{3}$$

where *A*, *B,* and *C* are constants, and *ʋ* is the average linear velocity (cm/s). The optimum value of *h* will be obtained when

$$\frac{dh}{dv} = 0$$

so that

$$v = \sqrt{\frac{B}{C}}$$

The term *A* is defined as the tortuosity factor, and it is affected by the size and the distribution of the stationary phase particles. The term *B* depends on the diffusion of the molecules in the longitudinal direction, linked to the motion of solute molecules in the mobile phase. This coefficient is significant at low flowrates. The term *C* refers to the resistance to the mass transfer of the analytes through the two phases and their diffusion on the surface. **Figure 1** presents a simplified van Deemter plot, which allows us to identify the optimum flowrate, corresponding to the minimum value of *h*.

In 1966, Pretorius and Smuts firstly observed that, using open tubular columns, it is possible to improve speed and efficiency of mass transfer with turbulent rather than laminar flow [16]. Turbulent flow in fact, dominated by rotational motion, reduces band-broadening, presenting a profile flatter than laminar flow (**Figure 2**).

**Figure 1.** *Simplified van Deemeter plot with A, B and C components.*

**Figure 2.** *a) Laminar flow; b) turbulent flow.*

*Turbulent Flow Chromatography: A Unique Two-Dimensional Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110427*

Physicist Osborne Reynolds found that the flow of a fluid through a straight and smooth tube transitions from laminar to turbulent as the momentum of the fluid becomes around two thousand times greater than its resistance to flow. The momentum, which is a certain amount of mass moving at a certain velocity, takes into account the fluid density and the diameter of the tube. It is possible to increase the fluid momentum by increasing its velocity, or by increasing the diameter of the tube, or both. The resistance to flow is expressed as the absolute or dynamic viscosity of the fluid [17].

The Reynolds number, Re, is a dimensionless parameter, defined as the ratio of the inertial to viscous forces present in a fluid system (Eq. 4).

$$\mathfrak{R} = \frac{\mu \mathbf{0} \ast I}{\eta} \tag{4}$$

where *μ<sup>0</sup>* is the mean linear velocity of the fluid, and *η* is the dynamic viscosity of the mobile phase. In a packed bed such as an HPLC system, the characteristic length scale *I* is related to particle diameter and external porosity.

The transition from laminar to turbulent flow occurs as Re overcomes some reference values. For a very straight and smooth cylinder the transition from laminar to turbulent flow occurs at a Reynolds number of 2400, while for a packed bed of uniform spheres, turbulence occurs between 1 and 10 Re [18]. Even if turbulent flow presents the advantage of better mass transfer, the backpressures required to obtain this flow would be excessively high, particularly in terms of pumping systems and particle architecture. In the 1990s, it was observed that using large irregularly shaped particles (50–100 μm), the backpressures were sufficiently low to generate turbulent flow and to improve column efficiency at increasing flowrate thanks to large interstitial spaces between particles (around 100 Å). In addition, it was observed that larger molecules in solution diffuse more slowly than smaller ones. Thus, larger molecules do not interact with the stationary phase, while smaller molecules have time to diffuse in and out of the pores and to interact with the stationary phase depending on their affinity. In 1997, these packed columns (now called TurboFlow™ columns) were patented (US patent no. 5,919,368) [19]. Although the mechanism is not fully understood, TurboFlow has demonstrated to be a very efficient method to separate large molecules, such as matrix components (e.g. proteins and lipids), from small molecules [14].

#### **2.1 Column chemistries**

TurboFlow™ columns are available in a wide variety of packing materials to enable different applications. The selection of the appropriate column must be based on the polarity of the analytes and the mobile phases needed to solubilize them. Initially, these columns presented an internal diameter of 1 mm and required a flowrate of 4–5 mL/min. Recent columns have an internal diameter of 0.5 mm to reduce solvent consumption and require a flowrate around 1.5–2 mL/min. It is possible to group TurboFlow™ columns in silica-based and polymer-based. Among silica-based columns, suitable for pH ranging from 2 to 9, packing includes Cyclone C18, Cyclone C8, and Cyclone C2 modified columns. Furthermore, Cyclone Fluro and Cyclone C18-P columns are also available. Styrene/divinylbenzene copolymers-based columns, suitable for pH ranging from 1 to 13, are also available in different chemistries: Cyclone, Cyclone-P, Cyclone mixed-mode with anion—(Cyclone MAX), cation—(Cyclone MCX), and mixed anion–cation-exchange phases (Cyclone MCX-2). Details about column applications are described in **Table 1** [20].


#### **Table 1.**

*Silica-based and polymer-based column applications.*

## **3. Hardware**

The turbulent flow chromatography, whose main peculiarity lies in the particular characteristics of its columns (described above), requires a specific hardware platform capable of exploiting the potential that these can express. The only instruments on the market designed to take full advantage of TurboFlow columns are marketed by Thermo Fisher Scientific as Transcend TLX systems.

These systems can be used as traditional HPLC, injecting the sample directly onto the analytical column, or for 2D-LC. In order to handle a turbulent flow chromatography and eventually to couple it with a second analytical column, a complex and unique hardware, which presents at least two pump systems and a valve interface module, is required. In addition, the systems can include multi-channel technologies. Depending on its configuration, each system can include one, two, or four pairs of pumps that enable the system to work either in single or 2D-LC mode over one, two, or four separate channels. When a sample is extracted/purified, the system uses one pump, named loading pump, to carry the sample into the TurboFlow column, and a second pump, the eluting pump, to resolve the extracts over a traditional analytical column. The overall plumbing of the system is intricate since it can include up to four pairs of valves each lodging a different couple of columns (the TurboFlow column in valve A and the analytical one in valve B) and, eventually, a special extraction loop, together with other valves for channel selection and for flow diversion to the detector or to the waste (**Figure 3**). Moreover, the system controls up to four couples of injection ports (for the online extraction and for the traditional elution) using one or two autosampler arms. Some interesting accessories can complete and further complexify the configuration, as for example a multicolumn compartment module (MCM) that allows to install simultaneously up to six extraction and six analytical

*Turbulent Flow Chromatography: A Unique Two-Dimensional Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110427*

#### **Figure 3.**

*Valve interface module of a) single channel, b) double channel, c) four channel systems. The "bypass" valve diverts the flow to the waste or to the detector.*

columns (or eleven columns of the same type) and to select the column to be used via software, without the need to plug and unplug them. Considering the complexity of Transcend systems, a dedicated software (Aria MX™) simplifies their use by controlling and coordinating the valve switching processes and the multiplexing capabilities.

Using Transcend systems for online extraction, two modes of operation are possible: Quick Elute Mode and Focus Mode (**Figures 4** and **5**). These differ in complexity, field of application and plumbing, being the Quick Elute mode simpler and faster.

Quick Elute is generally used for single compound quantification and/or when speed is the priority. In this case, in which only a TurboFlow column is needed, chromatographic separation is performed, while compounds are eluted from the column. This operating mode presents the advantages of being fast and simple to be set even when analysing complex matrices. Furthermore, it requires minimal method development. However, this separation presents a limited resolution, because of the large irregularly shaped particles of TurboFlow columns, so possible isobaric interferences should be taken into account. Nonetheless, it is possible to add an analytical column between the valve B and the detector for a rapid 2-D separation, similar to an online SPE, with the advantages that a turbulent flow gives to the extraction phase.

The Quick Elute Mode consists of the following steps: loading, eluting, column cleaning, and reconditioning. The sample is firstly injected into the loading pump mobile phase flow. The analytes are brought to and retained by the column, installed on the A valve, while the matrix macromolecules, not retained by the column, are washed to the waste connected in the B valve. If the analytes are not retained, they will be washed away in this step, if they are too retained, they will be washed out during the column cleaning process. After the loading step and before the elution, the TurboFlow column can be rinsed with a stronger mobile phase to purify the sample.

By switching the B valve, the mobile phase from the eluting pump flows through the TurboFlow column, the analytes are eluted to the detector or, if the case, to an analytical column. It is possible to elute the analytes off the TurboFlow column using

*Quick elute valve arrangement without analytical column.*

**Figure 5.** *Focus mode valve arrangement.*

#### *Turbulent Flow Chromatography: A Unique Two-Dimensional Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110427*

the same flow direction as the loading step (forward flush), or in the opposite direction (back flush), by switching the A valve. After the elution step, the extraction column is washed by the loading pump flow with an appropriate organic solvent. The re-conditioning presents the same parameters of the loading step, but for a longer time to better equilibrate the system.

When the quantification of multiple compounds is requested, Focus Mode is indispensable. It involves a TurboFlow and an analytical column and requires more extensive method development. In addition to the Quick Elute mode setup, the Focus Mode requires an elution loop in the A valve for the transfer step and a customized tee-piece rotor seal in the B valve (**Figure 6**).

The Focus Mode consists of six steps: loading, sample washing, transfer, system washing, loop-filling, and reconditioning (**Figure 7**) [20]. In the loading step, samples are injected into the TurboFlow column at high flow rates so that macromolecules of the matrix, salts and ionic compounds, not retained by the column, flow to waste (**Figure 7a**). Once loaded, the sample is cleaned from eventual contaminants using an appropriate mobile phase (**Figure 7b**). During these two steps, the mobile phase of the eluting component flushes to the analytical column connected to the detector.

During the transfer step, the valves A and B are switched, the optimized loop content is pushed by the mobile phase into the TurboFlow column, and the analytes are eluted off and transferred to the analytical column (**Figure 7c**). In this step, the transfer flow is diluted, via the tee-piece rotor seal, in the B valve, with the mobile phase coming from the eluting pump, in order to focus the molecules of interest to the head of the analytical column. These first three steps typically take around 2 minutes.

When the transfer is completed, the B valve is switched, the loading and eluting flows are again separated and, while the analytes are resolved on the analytical column and eluted to the detector, the TurboFlow column and the extraction components are washed to avoid carryover (**Figure 7d**). At this moment, the loop is filled with the proper transfer solution to be used in the following injection (**Figure 7e**). In the final step, the elution loop is isolated, and the columns are re-equilibrated to the conditions of the loading step (**Figure 7f**).

#### **3.1 Operations in multichannel systems**

In a traditional LC run, analytes of interest are usually eluted in a small portion of the entire chromatogram. It means that, during the pre-injection phase (needle and injector wash, sample withdrawn, etc.), but also during the first and last minutes of a run, the detector acquires useless data or the flow is diverted to waste. TurboFlow is multichannel systems, in which each "channel" is an independent HPLC equipment with its own pump(s) and injector port(s). These channels are connected to one single detector, and the systems are able to maximize the productivity, reducing the idle

**Figure 6.** *Rotor seals in a) quick elute mode, b) focus mode.*

**Figure 7.**

*a) Loading; b) sample washing; c) transfer; d) system washing; e) loop filling; f) reconditioning.*

time of the detector. Thanks to the multiplexing, in fact, TurboFlow staggers injections, by overlapping the channels to optimize the time of the acquisition window. In particular, two- or four-channel systems are currently available (**Figure 8**). Aria MX™ software is able to calculate automatically the timing of injections based on the relative duration of acquisition windows in comparison with the duration of the entire chromatogram and the pre- and post-injection operations of the auto-sampler. On each of

**Figure 8.** *Representation of staggered injections in a multiplexing system.*

*Turbulent Flow Chromatography: A Unique Two-Dimensional Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110427*

#### **Figure 9.**

*Diagram showing the setup of a two- (squared in purple) and a four-channel (squared in red) multiplexing.*

the different channels, the system is able to handle runs with the same or different instrumental method, maximizing the productivity up to four times when using a four-channel system. However, the hardware complexity is also significantly increased (**Figure 9**).

## **4. Development of a focus mode method**

The two approaches described above require different method development. However, considering the complexity of the Focus Mode, in the present paragraph only its method development will be explained.

A Focus Mode involves at least two columns, a TurboFlow and an analytical column. Two mobile phases (appropriately chosen) for the analytical gradient elution and at least two mobile phases for the extraction component for loading, transfer and washing steps are also requested. It is generally suggested to start with the choice of the ideal analytical column and elution conditions, able to chromatographically resolve the analytes. Then, considering the type of chromatography for the extraction process (i.e. reverse phase, ionic exchange, etc.) intended to perform, a TurboFlow column has to be chosen, compatible with the chemical characteristics of the analytes, the mobile phases, and the column selected for the analytical separation. Considering the possible combinations of couples of columns, a multiple column module (MCM) can help in this evaluation. Once the separation conditions are set, loading, transfer and washing conditions have to be optimized. This process that can be facilitated by the Aria software, consists of studying the analytes chromatographic behaviour when mobile phase composition, duration, and flowrate are changed during the different steps. In fact, one of the software useful features allows one to test different conditions for a specific method parameter, defining it as "variable" and programming its values in the acquisition sequence instead of writing one method per condition that has to be tested. When optimizing these parameters, the analytical column has to be

removed. A T-union, which splits the flow of the eluting mobile phase between detector and waste, is connected to detect the analytes elution at all steps in the method (**Figures 7** and **10**).

During the optimization of the loading and washing step conditions in a classic TurboFlow method, in which reverse phase chromatography for analytes extraction is used, different percentages of organic solvent have to be tested to establish the highest % of organic phase able to wash the sample without eluting analytes to the waste. In the wash step all interferences more polar than the molecules of interest are ideally washed out. In the example reported in **Figure 11**, tetrahydrocannabinol (THC) was loaded in a Cyclone-P column in 100% aqueous phase and an increasing percentage of organic phase was tested for the washing step. The ideal condition of the washing step

#### **Figure 10.**

*Method development setup: The analytical column is replaced by a T-union diverting only the proper flow from the TX column to the MS detector.*


*Optimization of loading conditions for the analysis of THC in a cyclone-P column. The percentages refer to the organic phase.*

#### *Turbulent Flow Chromatography: A Unique Two-Dimensional Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110427*

was at 30% of B (**Figure 11**). Results with a higher organic content (40% and 50% in **Figure 11**) showed peaks appearing during the wash step, therefore demonstrating that the analytes are no longer retained on the TurboFlow column. Some loss during the loading/washing steps might be accepted as long as this is not impacting the required limits of quantification, since this will make the sample cleaning more efficient.

In the Transfer step, the analytes elute from the TurboFlow column and are transferred to the analytical column by the contents of the transfer loop. The solvent strength of the loop content is diluted before entering the analytical column by the eluting pump flow. The goal in this step is to be able to quantitatively elute the analytes from the TurboFlow column and to focus them on the head of the analytical column while reducing as much as possible the hydrophobic interferences from the sample matrix (matrix effect). So, three development stages are required: optimization of the transfer loop content, review of the transfer time using different transfer flowrates, and finally optimization of the transfer step dilution ratio.

Regarding the transfer loop content, the goal at this stage is to determine the minimum amount of organic solvent that quantitatively transfers all the target analytes to the analytical column, without eluting more hydrophobic compounds. The lower the content of organic solvent in the loop, the higher the retention of late eluting components (e.g. phospholipids) in the TurboFlow column (then washed to waste during the wash step), reducing matrix effect during the chromatographic elution.

If the solvent strength of the loop is too low, the analytes remain retained in the TurboFlow column and will be washed to waste during the washing step. On the other hand, if the solvent strength is too high, focusing of the analytes on top of the analytical column might be affected, leading to chromatographic peaks distortion or lack of retention. Moreover, the purification process would be less efficient. During the method development, the optimization of the loop content is performed without the analytical column.

In the example of the method development for the analysis of THC in the Cyclone-P column, decreasing percentages of organic phase were tested, and the ideal one was identified as 50% (**Figure 12**). Indeed, for lower content of organic solvent in the loop, part of the analyte was not transferred and was washed out in the washing step.

It is advisable to evaluate the transfer step time for different loading pump flowrates in preparation for the next step of the method development, the dilution ratio. If the flowrate of the loading pump during the transfer step is high, the method will be faster. However, the dilution from the eluting pump will be lower (taking into account that the sum of loading plus eluting flow cannot exceed the flowrate suitable for the analytical column), and hence, it will be more difficult to focus the target analytes on the head of the analytical column. This evaluation step consists in recording the time it takes for the analytes to reach the detector as the eluting flow varies in the transfer step. For the transfer time evaluation, there are no right or wrong values, but the information about the transfer that will be used in the final method, once the dilution ratio will be established, is just collected.

After the installation of the analytical column, the dilution ratio can be assessed, keeping the goal to focus the extracted analytes on the analytical column. If the organic concentration of the mobile phase in the transfer step is too high when it enters the analytical column, the analytes move through the analytical column rather than focus on it. In order to reduce this effect, the eluting pump dilutes the organic mobile phase from the loop with aqueous mobile phase reducing the solvent strength of the combined flow into the analytical column. The final solvent strength is

**Figure 12.**

*Optimization of loop fill content for the analysis of THC in a cyclone-P column. The percentages refer to the organic phase.*

influenced by the organic concentration of the loop contents and of the eluting pump flow (already optimized in the previous steps) and by the relative ratio between the eluting and loading pump flowrates (Eq. 5).

$$\text{Final\%} \text{or} \text{gains} \text{solvent} = \left(\frac{\text{LP}}{T}\right) \* \text{OL} + \left(\frac{\text{EP}}{T}\right) \* \text{OEP} \tag{5}$$

where T is the total flowrate, the combined flowrate of the loading and eluting pumps during the Transfer step; LP is the loading pump flowrate during the transfer step; EP is the eluting pump flowrate during the transfer step; OL is the organic content percentage in the loop, and OEP is the organic percentage in the eluting pump flow.

Thus, the transfer dilution ratio has to be optimized by testing the effect of different combinations of loading and eluting flowrates on chromatography, taking into consideration that the total flowrate should match the flowrate used for the analytical column.

Peak fronting, poor resolution, and breakthrough in the final chromatography could be observed if solvent strength is too high during transfer and should be fixed trying to reduce the overall organic content during the transfer step.

Once the method is optimized, the maximum injection volume, which follows the same rules of a regular liquid chromatography method, has to be evaluated. The injectable sample volume will be higher when its composition is more similar to the

*Turbulent Flow Chromatography: A Unique Two-Dimensional Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110427*

**Figure 13.** *Maximum injection volume identification.*

initial mobile phase composition. However, it has to be considered that turbulent flow, compared to laminar flow, allows the use of a higher percentage of organic solvent. Increasing injection volumes of an extracted matrix sample, correlated to proportional increase of peak area, should be tested. The maximum injection volume corresponds to the volume that causes a flattened out peak area. The example in **Figure 13** shows that the maximum injection volume is 75 μL. Nonetheless, thanks to high carbon load values, the columns capabilities usually can handle volumes of 100 μL, significantly improving the overall method sensitivity, compared with injection volumes of conventional methods (5–10 μL).

## **5. Applications**

Successful applications of the turbulent flow technique have been reported in different fields, such as therapeutic drug monitoring and environmental analysis, applied to various matrices, such as urine, plasma, but also food materials and water (**Table 2**).

Most of the published papers are focused on clinic applications. In this field, sample pretreatment, separation, and detection need to be more integrated in order to make mass spectrometry routinary even for not specialized laboratories. Online multidimensional chromatography, like TurboFlow methods, combining sample preparation and analysis, facilitate sample introduction, ease-of-use, and speed, even when dealing with complex matrices. Turbulent flow chromatography allows a simplified analysis of plasma, serum, or whole blood [21–26]. Hervious and colleagues reported the development, validation, and application of LC–MS/MS coupled with TurboFlow for the quantification of irinotecan, a cytotoxic agent used for metastatic


*Turbulent Flow Chromatography: A Unique Two-Dimensional Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110427*



**Table 2.**

*Some applications of turbulent flow chromatography reported in literature.*

colorectal cancer treatment, and its active and inactive metabolites, SN38 and SN38-G, respectively, in plasma after protein precipitation [27]. The same approach was applied for human plasma screening of various drugs that could be identified also below the therapeutic concentration [28]. Therapeutic drug monitoring of tyrosine kinase inhibitors (TKIs) is crucial for various cancers treatment. Early analytical methods involved HPLC-UV. However, not all TKIs present a good UV absorbance. TurboFlow LC–MS/MS was successfully used for the quantification of these compounds in a single analysis. In this protocol development, a precipitation step before online extraction was proven to maximize column life-time and minimize risk of autosampler blockage [14]. Multiplexed, multi-dimensional uHPLC–MS/MS was applied not only to the analysis of human plasma, but also to the high-throughput screening of lysosomal storage disorders in newborn dried bloodspots to obtain an online sample clean-up [29]. Determination of contaminants in urine, such as Bisphenol A and other environmental phenols [30], pesticides and metabolites [31, 32] or toxins [33] have been reported. Turbulent flow chromatography finds its application also in forensic toxicology. Mueller and colleagues developed a fully automated toxicological screening system for online urine extraction and analysis [48]. In the context of driving ability diagnostic, TurboFlow methods for the quantification of opiates, cocaine, amphetamines, methadone, and benzodiazepines have been validated in urine matrices [34].

Although the applications in drug monitoring are predominant, the use of TurboFlow is also reported in food and environmental quality. In fact, food products contain analytes in low levels, and other constituents, potentially interferents, such as sugars, proteins, and pigments in significantly higher concentrations, determining the necessity of sample clean-up and/or pre-concentration. The applicability of this technique in food analysis has already been proved in the determination of Fusarium mycotoxins [35] and of plant and fungal metabolites in wheat, maize, and animal feed [36], but also in the determination of melamine in infant formula [37]. A successful method has been developed and applied by Mottier and colleagues for the determination of 16 fluoroquinolones in honey, used to treat bee's bacterial diseases [38]. This technique allowed the reduction of extraction time and elimination of interferences in complex samples such as royal jelly and the determination of polyphenols [39]. A TurboFlow method was validated for the quantification of flavonoids and resveratrol in different types of wine. The authors suggested applying the method for the quantification of flavonoids, which could be correlated to the type of pesticides and of grapes

#### *Turbulent Flow Chromatography: A Unique Two-Dimensional Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110427*

used to produce wine [40]. Online purification based on turbulent flow chromatography for the simultaneous quantification of multiple pesticides residues is reported in different matrices, such as Chinese cabbage and cucumber samples [41], grape, wheat flour, and carrot-based puree baby food [42]. Veterinary drug residues of 36 antibiotics from seven different chemical classes were identified and quantified in chicken meat, bringing the benefits of automation and cost-effectiveness [43]. The versatility in the analysis of different matrices has been confirmed in a study comparing conventional and online sample clean-up system for the determination of deoxynivalenol and its conjugated derivative, deoxynivalenol-3-glucoside, in cereal grains [44].

Recently, laboratories have begun to move towards alternative and greener methods for environmental analysis, in response to the growing awareness of more sustainable and environmentally friendly techniques. Online sample pretreatment responds to this trend, providing the development of effective approaches with low or no solvent and chemical consumption for the analysis of trace contaminants, such as pesticides. A TurboFlow method, for example, was successfully applied to the analysis of a wide spectrum of trace level pesticides, in drinking and surface water samples, taken from several sampling sites. Different TurboFlow columns were tested, showing that polymer-based columns offered the best performance [45, 46].

Pharmaceuticals are other relevant environmental trace contaminants. Their ecotoxicology is well known, while less is known about their metabolites and transformation products effects. Thus, multi-residues analytical methods are crucial to assess the risk of their presence in the environment. Furthermore, due to their low concentration, a pre-concentration procedure is often mandatory. Lopez-Serna and coworkers presented the development of an efficacious method for 58 pharmaceuticals and 19 metabolites/transformation products with an online pre-treatment based on turbo-flow chromatography in environmental aqueous samples. The combination of three TurboFlow columns in sequence showed the highest sensitivity [47].

## **6. Conclusion**

Turbulent flow chromatography is a useful technique, able to remove potential interferences and to reduce preparation steps in an efficient way. In addition, the multiplex system allows to obtain faster results, by switching between different methods with minimum manpower, enhancing laboratory high-throughput productivity. Even if potentially heavy-matrix samples, as biological ones, could be directly injected into the TurboFlow column without any pre-purification, when performing quantitative analysis, in which the use of an internal standard is advisable, a simple addition of an organic solution of the internal standard to the samples could be performed, partially precipitating the protein content of the solution to be injected. In this way, the lifetime of the columns would be maximized. The resistance of the columns, granting hundreds of injections, and the possibility to integrate the TurboFlow to a pre-existent mass spectrometer, whose sensitivity is improved, guarantees also cost-effectiveness. Nonetheless, a high hardware and method development complexity have to be considered. This approach has only lately been increasing its applications, probably due to the complexity of the analytical methods development and optimization. Clinic and toxicology, where mass spectrometry must be handled even by not specialized laboratories, are the fields in which TurboFlow finds major application. However, TFC started to find use in food and environmental quality control, and it is intended to expand in these fields in the future.

## **Acknowledgements**

The authors would like to thank Dr. Mariana Barcenas Rodriguez (European Molecular Biology Laboratory, Germany), Pr. Serena Indelicato (University of Palermo, Italy), and Pr. David Bongiorno (University of Palermo, Italy) for their advices and suggestions during the writing of this chapter and Dr. Claudio De Nardi (Thermo Fisher Scientific, U.S.), the most skilled TurboFlow user we know, for introducing us to this powerful system.

## **Author details**

Francesca Di Gaudio<sup>1</sup> , Annamaria Cucina<sup>2</sup> \* and Sergio Indelicato<sup>2</sup>

1 PROMISE-Promotion of Health, Maternal-Childhood, Internal and Specialized Medicine of Excellence "G. D'Alessandro", Palermo, Italy

2 Chromatography and Mass Spectrometry Section, Quality Control and Chemical Risk (CQRC), Ospedali Riuniti Villa Sofia – Cervello, Palermo, Italy

\*Address all correspondence to: annamariacucina1@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Turbulent Flow Chromatography: A Unique Two-Dimensional Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.110427*

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## **Chapter 3**

## Monoliths Media: Stationary Phases and Nanoparticles

*Mohamed Hefnawy, Ali El Gamal and Manal El-Gendy*

## **Abstract**

Monoliths media are gaining interest as excellent substitutes to conventional particle-packed columns. Monolithic columns show higher permeability and lower flow resistance than conventional liquid chromatography columns, providing highthroughput performance, resolution and separation in short run times. Monolithic columns with smaller inner diameter and specific selectivity to peptides or enantiomers have been played important role in hyphenated system. Monolithic stationary phases possess great efficiency, resolution, selectivity and sensitivity in the separation of complex biological samples, such as the complex mixtures of peptides for proteome analysis. The separation of complicated biological samples using columns is being revolutionized by new technologies for creating monolithic stationary phases. These techniques using porous monoliths offer several advantages, including miniaturization and on-line coupling with analytical instruments. Moreover, monoliths are the best support media for imprinting template-specific sites, resulting in the so-called molecularly-imprinted monoliths, which have an extremely high selectivity. In this chapter, the origin of the concept, the differences between their characteristics and those of traditional packings, their advantages and drawbacks, theory of separations, the methods for the monoliths preparation of different forms, nanoparticle monoliths and metal-organic framework are discussed. Two application areas of monolithic metal-organic framework and nanoparticle monoliths are provided.

**Keywords:** monoliths media, advantages of monoliths, hybrid monoliths, nanoparticle monoliths, monolithic metal–organic framework

## **1. Introduction**

Liquid chromatography is a separation technique that uses a fluid phase through a porous bed. In order for the sample components to quickly equilibrate between the fluid and solid phases and spend a sizable portion of their entire residence durations in both, the fluid and bed materials must be selected carefully. The core of chromatography is a porous bed because the percolation of the mobile through the stationary phase is a crucial aspect of the chromatographic procedure. Solute molecule exchanges between the mobile phase stream and the stationary phase should occur quickly and often. Fast mass transfer kinetics, diffusion, close spacing within the bed, and a sizable surface area of contact between the stationary and mobile phases are all desirable

characteristics for the column bed. High column efficiency is required if adsorption serves as the retention mechanism [1].

However, the bed's hydraulic resistance to the stream of the mobile phase should be kept at a reasonable level. The maximum length of the column that can be used with a specific pumping system will be impacted by a high hydraulic resistance. Fine particles should enhance separations in the early stages [2]. Unfortunately, there is not much that can be done to improve a packed bed's permeability. This permeability is determined by the packing density of the particles, which is best determined by the external porosity of the bed, and by their average size. With rising porosity, the permeability rises quickly in almost perfect proportion to its fifth power [3].

Now, imagine a porous bed that is the converse of a packed column bed. All the parts of the packed column bed result in a slower mobile phase flow velocity. The trend, so far, has been to prepare columns that have a high volume of through pores, hence a low hydraulic resistance leading to increase velocity. This makes it possible to prepare columns that have a high efficiency, because they can be long and yet can be operated with an acceptable head pressure, and are relatively fast, because they are operated at high flow velocity. In actual practice, a decrease in the column inlet pressure is far less attractive than a decrease in the elution time of the last eluted sample component or an increase in the column efficiency [4].

The popular English dictionary defines the monolith as a geological or technological feature such as a mountain or possibly a boulder, consisting of a single massive stone or rock [5]. Erosion usually exposes these formations, which are most often made of very hard and solid metamorphic rock. However, large pieces of rocks excavated from quarries, such as obelisks, are called artificial monoliths (**Figure 1**).

**Figure 1.** *Photograph of the porous monolith erected at the entrance of the Summer Palace Park, Beijing, China (Reprinted from Ref. [6]).*

## **2. Monoliths**

### **2.1 Definition**

Monolith, in its broadest sense, refers to a column made out of a single substantial stone block. The name comes from the Greek words monos, which means "single," and lithos, which means "stone." It resembles a continuous single rod of porous material in chromatographic terms [7].

## **2.2 Advantages of monoliths**

Due to the homogeneous distribution of macropores and mesopores across the network, which enables the separation of numerous analytes, monoliths are distinguished by a high permeability. While mesopores offer a significant surface area for separation and the permeability for solvent flow, macropores allow 100% of the mobile phase to pass through the column. In addition, packing like in a particlepacked column is not essential because the monolith can be created in place using poly-mediation. Due to the issue of the monolith shrinking in capillaries with greater i. d., this polymerization technique is only permitted in capillaries (typically less than 200 m in internal diameter i.d.). The distinctive characteristics of monolith, which have drawn the attention of numerous researchers and have been demonstrated to be an effective instrument in column technology [8], are high porosity and low back pressure. Pioneer efforts by Hjerten *et al.* [9] and Svec and Frechet [10] on developing polymer monolith and later by Minakuchi *et al.* [11] and Ishizuka *et al*. [12] developed silicon monolith have revolutionized the field of column technology.

Initially restricted to academic research, monolith is now acknowledged as a legitimate member of stationary phases. Other than permeability, monolithic columns are similar to conventional columns in terms of phase ratio and enantioselectivity [13]. The preparation and modification of monolithic columns to the correct porosity and pore diameter to meet various purposes are also simpler [14]. Through co-polymerization, specific selectors, such as chiral selectors, can be integrated into the monoliths and preserved there. In comparison to a particle column, the elution time can be cut by a factor of 5 to 10 [15]. All of these processes do not require any specialized knowledge, which makes interlaboratory investigations simple and comparable [13].

### **2.3 General characteristics of monoliths**

Monolithic columns are made of a single piece of porous material that is hermetically sealed against the tube wall. As a result, the mobile phase stream is unable to bypass any appreciable portion of the bed and must instead percolate through it. A bimodal pore size distribution of the porous material is frequently used to describe it. This distribution's huge size mode corresponds to through pores or macropores. These pores are connecting into the channels that carry the majority, if not nearly all, of the mobile phase. In order to maintain the correct flow rate, the mobile phase must be pushed into the column at a head pressure determined by the average size of these channels, which also determines the column's permeability. Concerning the connection of the channels generated by these *via* pores, their constriction, and column permeability. The mesopore structure of the proton's polarons and all of these variables work together to determine the kinetics of mass transfer and column efficiency.

Practically no small size mode can be seen in the pore size distribution of the majority of polymeric materials [16–18].

Mesopores are represented by the small size mode of the pore size distribution of inorganic monoliths, such as silica. Between the channels that the through pores have created, there are lumps of porous material that contain these mesopores. For lack of a better term, these lumps are referred to as "porons." The lumps of porous material between the larger holes of particles with a bimodal internal porosity were first given this term [6]. The total porosity of the bed and its mechanical stability (the bed must withstand the pressure drop applied to cause the mobile phase to percolate through it) as well as the internal porosity and the precise surface area of contact between the two phases of the chromatographic system are correlated with one another. The latter regulates the solutes' retention factors and phase ratio. Therefore, the internal porosity of the column must strike a balance between the demands for small porons, a significant portion of the column's volume occupied by these porons, and a relatively large average size of the through pores [6]. More than two forms of pore size distribution may exist in monolithic materials. Because of their high adsorption energy and delayed mass transfer kinetics, micropores in any adsorbent have consistently had a negative impact on chromatographic performance. In polymer matrices and with silica-based adsorbents, microspores are uncommon [19], although they are prevalent in carbon-based adsorbents [20]. Another option is the mode associated with pores that have an average size that falls somewhere between the diameters of mesopores and through pores. This would be related to a certain class of monoliths with geometrical properties resembling those of the particles with bimodal porosity that were promoted for perfusion chromatography [21].

#### *2.3.1 The through pores*

Numerous research teams, including those of Nakanishi [22–24], Tanaka *et al*. [25], and later Cabrera [26, 27] and Leinweber [28], have explored the porous architectures of silica monoliths in depth. Phase separation is a byproduct of the acidcatalyzed hydrolysis and gelation of an alkoxysilane solution (such as tetramethoxysilane) in the presence of sodium polystyrene sulfonate, as demonstrated by Nakanishi and Soga [29]. Then, these researchers were able to create a solid mass of porous silica with a bimodal porosity by switching polyacrylic acid (PAA) with polystyrene sulfonate [22, 23]. Later, HPAA was effectively used to replace polyethylene oxide (PEO) [30]. Chromolith silica rod, a commercial item from Merck KGaA (Darmstadt, Germany), is depicted in **Figure 2** as having a porous structure [27]. By varying the solvent composition, the concentration of porogen, and the temperature, it is possible to modify the gel shape. The average size of the through holes grows with increasing porogen concentration (for example, HPAA). At low porogen concentrations, the big pores are not linked, whereas at high concentrations, distinct particles form [22]. Mercury intrusion porosimetry was used to estimate the average size of the through pores, which was found to be close to 1.7 m, and the external porosity or porosity of the through pores, which was calculated at 0.65 m [31].

#### *2.3.2 The porons*

The through pore network and the entire column of porons are filled with a continuous porous solid called porons. The monolith made using the method described by Nakanishi *et al*. [22, 24, 29] and bound to octadecyl dimethyl silyl groups

**Figure 2.**

*Image of the porous structure of a typical monolithic silica column (left) and enlarged view of the entrance to a macropore or through pore (right) (Reprinted from Ref. [27]).*

was shown by Nakanishi *et al*. [24] to be a porous solid with bimodal porosity. It was discovered that the porons had an average size of roughly 1 m. For neat silica, the mesopores or internal porosity are about 0.20 m [32].

## **3. Theory of separation**

The most important characteristics of any chromatographic column are its permeability, which is determined by the average size of its through pores and external porosity, its efficiency, which is determined by the average sizes of its through pores and porons, the structure of the mesopore network, and the mobile phase velocity, and its capacity for retention (that is related to the specific surface area of the adsorbent, to the internal porosity and the pore size distribution). The monolithic beds prepared for liquid chromatography and the relationships between the characteristics of monolith beds and the permeability and efficiency of the prepared columns. Finally, the characterization of the monolithic columns and the conventional packed columns is used to compare these columns [33].

## **4. Separation mechanism**

Hydrophilic interaction is hypothesized to be the main mechanism of hydrophilic interaction liquid chromatography (HILIC) separation for neutral polar molecules. The impact of the organic solvent concentration (mostly, acetonitrile, ACN) on the retention of small compounds (such as toluene, thiourea, and acrylamide) has frequently been studied in order to assess the HILIC capabilities of new stationary phases. The retention of polar molecules increases with increasing organic solvent content in HILIC stationary phases, which often exhibit typical HILIC behavior at high organic solvent content before returning to an apparently reversed phase (RP). When the stationary phases contain some hydrophobic characteristics and the organic solvent, content is below a threshold composition. In order to determine the polarity of HILIC stationary phases, one can use the critical composition of the mobile phase,

which corresponds to the change from the HILIC to the RP mode [34]. The critical composition typically transitions to a lower concentration of ACN in the mobile phase with more polar stationary phases, permitting the use of mobile phases with higher water content in the HILIC mode. Porous zwitterionic monolithic columns exhibited very high selectivities for polar analytes, particularly tiny peptides, it was claimed [35–38]. Electrostatic repulsion-hydrophilic interaction chromatography was a new phrase proposed by Alpert [39] (ERLIC). Unique selectivity for charged polar analytes is provided by the interaction between hydrophilic contact and electrostatic repulsion. Numerous HILIC monolithic columns have shown similar ERLIC behavior. Ibrahim *et al*. [40] examined the impact of mobile phase salt concentration on the retention of two amino acids on coated silica monolith. Improving the quantity of methyl phosphonate decreases the electrostatic attraction between the cationic analyte and the cationic latexes, increasing the retention of the basic amino acid histidine. For the acidic amino acid, aspartic acid, however, raising the quantity of methyl phosphonate shields the electrostatic attraction, lowering its retention. The monolithic columns built with acrylamide also included an intriguing mixed-mode separation mechanism. A number of polar aromatic compounds were often used to analyze these relatively polar stationary phases [41]. The retention factors of the better-retained compounds were drastically reduced by a rise in mobile phase polarity, as would be expected for HILIC. However, some elution orders also changed, indicating that other mechanisms besides simple hydrophobic changes may be at play. This suggests that the elution order was not merely the inverted form of the reversed-phase separation [42]. It was reported that polar and charged analytes were retained by a mixed mode of mechanisms, including hydrophilic interaction, electrostatic interaction, H-bridging interaction, interaction, and possible aromatic adsorption [43, 44].

Similar to HPLC, the mechanism of mass transfer involves hydrophobic adsorption on the sorbent's surface and partition into bound alkyl chains. When transferring analytes to the adsorption process, physical parameters (such as surface area, average pore diameter, and pore volume of the adsorbents) are also taken into account. In general, the surface area of the adsorbent has a direct relationship with its adsorption capacity. The molecules adsorbed in the diffusion process have a relationship with the pore size. The saturation capacity of monolithic and packed silica columns was compared *via* frontal analysis for four distinct compounds, and the results showed that the monolithic columns had a 30–40% greater capacity while the binding constants were almost identical for both [45].

This shows that even while the chemistry of both surfaces is extremely similar, resulting in the same elution order for many analytes, the monolithic column's efficacy or accessible surface area is greater than that of the packed column. However, a conventional column of the same size has a far better load capacity since monolithic columns have a much lower density. In other words, within a certain velocity range, the shape of the breakthrough behavior of monoliths (BTCs) does not change. This indicates a very fast mass transfer and allows the speculation that the mass transfer is not dominated by the chromatographic velocity [45].

BTC shape is determined by equilibrium isotherm, mass transfer resistances, and axial dispersion [45].

Because the flow pattern can be represented as an axial dispersion plug flow, the differential fluid phase mass balance is:

$$-D\_L \,\mathrm{\delta}^2 \,\mathrm{c} / \delta \mathrm{z}^2 + \mathfrak{u} \,\left( \mathrm{\delta} \mathbf{c} / \delta \mathbf{z} + \delta \mathbf{c} / \delta \mathbf{t} \right) + \left( \mathbf{1} - \dot{\mathfrak{e}} / \dot{\mathfrak{e}} \right) \delta \mathbf{q} / \delta \mathbf{t} = \mathbf{0} \tag{1}$$

with *DL* the axial dispersion coefficient, *q* the sorbate concentration averaged over the particle, *c* the fluid phase concentration, *z* the column length, u the interstitial fluid velocity, *t* the time, and *ἑ* the voidage of the adsorbent bed.

The mass balance for an adsorbent particle yields the adsorption rate expression, which may be written as.

$$\mathbf{\color{red}{6q/\delta t = f\ (q, c)}}\tag{2}$$

The global mobile phase power model, apparent solid phase power model, or twofilm model were all used to solve this equation. The mobility phase apparent total driving force model can be used to explain the mass balance of the adsorbed particle.

$$
\delta \mathbf{q} / \delta \mathbf{t} = \mathbf{6} / \mathbf{d}\_{\mathbf{p}} \, \mathbf{K}\_{\mathbf{c}} \, (\mathbf{c} \mathbf{-c}^\*) \tag{3}
$$

or the apparent overall solid-phase driving force model.

$$\delta \mathbf{q}/\delta \mathbf{t} = \mathbf{6}/\mathbf{d}\_{\mathbf{p}} \, \mathbf{K}\_{\mathbf{q}} \left(\mathbf{q}^\* - \mathbf{q}\right) \tag{4}$$

where c\* is a fictitious concentration in the mobile phase that is in equilibrium with q, and q\* is a fictitious concentration in the solid phase that is in equilibrium with c. The apparent total mass-transfer coefficients are Kc and Kq, respectively, while the surface-to-volume ratio for spherical particles is 6/dp. (c - c\*) and (q\* - q) are the apparent total driving forces, respectively. The overall apparent driving forces are, respectively, (c - c\*) and (q\* - q) [45].

The linear driving forces in the two-film theory are (c - c0) and (q0 - q), where subscript 0 designates a concentration at the two films' interface at which c0 and q0 are by definition in equilibrium, that is.

$$\mathbf{\dot{\sigma}} \mathbf{\dot{q}} / \mathbf{\dot{\sigma}} = \mathbf{\dot{\sigma}} / \mathbf{d\_p} \text{ } \mathbf{K\_f} \text{ (}\mathbf{c} - \mathbf{c\_0}\text{)} = \mathbf{\dot{\sigma}} / \mathbf{d\_p} \text{ } \mathbf{K\_s} \text{ (}\mathbf{q\_0} - \mathbf{q}\text{)}\tag{5}$$

where kf is a velocity-dependent external mass-transfer coefficient and ks is the solid-phase mass-transfer coefficient. 1/kf and 1/ks are termed the external and solidphase resistances.

Analytical formulas for the BTC can be established for various limiting forms of the isotherm, such as linear or rectangular, and axial dispersion is ignored.

BTCs can be predicted appropriately assuming that the equilibrium isotherm is rectangular when the actual equilibrium constant is greater than five [45].

At constant pattern, the mobile and solid-phase concentrations are related by Eq. (6).

$$\mathbf{q}/\mathbf{q}\_{\mathrm{F}} = \mathbf{c}/\mathbf{c}\_{\mathrm{F}} \tag{6}$$

where qF is the appropriate equilibrium concentration and cF is the feed concentration. The flow equations are transformed into ordinary differential equations under circumstances of the constant pattern. The constant pattern relation, Eq. (6), is changed to y = x because it is convenient to add the dimensionless concentrations x = c / cF and y = q / qF. The flow can be represented as a function of x by inserting Eq. (6) into Eqs. (3) and (4) and calculating c\* and q\*.

$$\mathbf{dq}/\mathbf{dt} = \mathbf{q}\_{\text{F}} \,\, \mathbf{dx}/\mathbf{dt} = \mathbf{6}/\mathbf{d}\_{\text{p}} \,\, \mathbf{K}\_{\text{c}} \,\, \mathbf{c}\_{\text{F}} \,\, \mathfrak{P}\mathbf{x}(1-\mathbf{x})/\mathbf{1} + \mathfrak{P}(\mathbf{1}-\mathbf{x})\tag{7}$$

and

$$\mathbf{dq}/\mathbf{dt} = \mathbf{q}\_{\text{F}} \,\, \mathbf{dx}/\mathbf{dt} = \mathbf{6}/\mathbf{d}\_{\text{p}} \,\, \mathbf{K}\_{\text{q}} \,\, \mathbf{q}\_{\text{F}} \,\, \beta \mathbf{x} (\mathbf{1} - \mathbf{x})/\mathbf{1} + \beta \mathbf{x} \tag{8}$$

where β = bcF.

Inserting the Langmuir expression and the constant pattern relation in the twofilm model, Eq. (5), yields.

$$\mathbf{\dot{s}}(\mathbf{x} - \mathbf{x}\_0) = (\mathbf{1} + \boldsymbol{\beta})\mathbf{x}\_0/\mathbf{1} + \boldsymbol{\beta}\mathbf{x}\_0 - \mathbf{x} \tag{9}$$

where δ is the scaled mass-transfer resistance ratio.

$$\mathbf{\dot{\delta}} = \mathbf{k\_{f}} \,\, \mathbf{c\_{F}} / \mathbf{k\_{s}} \,\, \mathbf{q\_{F}} \tag{10}$$

The interfacial mobile phase concentration without dimension x0 is obtained by solving Eq. (9), which in this instance may be done analytically [45]. The flux in the two-film model is expressed as a function of x by solving Eq. (9) for x0 and inserting it in Eq. (5). The solution is.

$$\mathbf{dq/dt} = \mathbf{q\_{F}} \,\mathrm{d}\mathbf{x/dt} = \mathbf{6/d\_{p}} \,\mathrm{k\_{f}} \,\mathrm{c\_{f}} \,(\mathbf{x-x\_{0}}) \tag{11}$$

Despite having identical properties (i.e., particle sizes of less than 2 m), monolithic silica adsorbs the analyte with the narrow band on the column because its back pressure is significantly lower than that of the particle sorbent [46]. In contrast, the analyte adsorbed is quickly eluted by a little volume of elution solvent since the analyte is concentrated in the column. In order to provide adequate sample capacity and extraction efficiency, the standard solid-phase extraction cartridge often needs an elution volume that is two or three times the volume of the sorbent bed. Monolithic silica, on the other hand, only requires 100 L of eluent to elute the analyte adsorbed [46].

## **5. Types of monoliths**

Based on the type of materials used in their preparation, monoliths are broadly categorized. Numerous monolith kinds may exist depending on this, although they are often divided into organic and inorganic-based monoliths. The chemistry of these two forms of monoliths serves as the basis for all other monolith types, whether through specific alterations or the use of many monomers. Organic monomers, like acrylamide [47, 48] and methacrylates [49, 50], are used for organic monoliths [51, 52], whereas inorganic monomers, like alkoxides of silicon [53, 54] are used for inorganic monoliths. The two categories differ in their chemistry of preparation, in which polymerization is applied for the organic monolith and hydrolytic polycondensation for the inorganic monolith.

#### **5.1 Organic polymer monoliths**

The idea of a monolith is thought to have originated from organic monoliths, which are composed of organic polymers. Developments in column technology have drawn a lot of interest since Hjerten *et al*. [9] introduced the first polymer monolith. His innovative work transformed the field of chromatographies by giving separating media new meaning and opening up new possibilities. They are a promising tool for separation in HPLC and capillary electrochromatography (CEC) modes due to the

### *Monoliths Media: Stationary Phases and Nanoparticles DOI: http://dx.doi.org/10.5772/intechopen.110502*

ease of fabrication, high permeability, porosity, and their ability to function in a wide pH range. They can be made using a variety of techniques, like radical polymerization (thermal or photoinduced) [55, 56], polycondensation reaction [57], and ring-opening metathesis polymerization [58, 59] depending on the type of stationary phase. Since the invention of the organic monolith, plenty of work has been carried out and it has been extensively reviewed. A mixture of monomers, a porogenic solvent, and an initiator are added to the chromatographic column or capillary to create polymerbased monoliths there. The final step is to treat the entire column or capillary to heator UV-initiated polymerization [60]. The initiator, which starts the polymerization of monomers, is converted into radicals. The leftover monomers and solvating solvents are distributed to the nuclei (precipitates) that the developing polymers create. The nuclei get bigger as the polymerization process goes on, which leads to clusters coalescing to produce a homogeneous integrated structure [61]. The monolith, which are uniform gels or stiff rods, is made up of interconnected clusters of globules [62]. The manufacture of organic polymer-based monolith typically involves the use of tubing with 8 mm i.d. capillaries for HPLC and 20–500 m i.d. capillaries for micro LC and CEC. Teledyne Isco, Inc. offers polymer monolithic columns for sale (IscoSwift®) [61]. Methacrylates, acrylamides, and styrene are frequently utilized as the monomers in organic monoliths [20]. Methacrylates monoliths have been thoroughly studied by Svec and his colleagues [55, 63]. Polystyrene was used to create a monolith by Wang et al. Several scientists then replicated their method to create the monolithic columns in a capillary style. The monolithic poly (polyphenyl acrylate-co-1,4-phenylene diacrylate) capillary columns were created by Bisjak *et al*. [64], and they investigated the impact of different polymerization parameters on the effectiveness of the separation. The monolith was created by thermally inducing the co-polymerization of phenyl acrylate and 1,4-phenylenediacrylate in the presence of azoisobutyronitrile, a radical initiator. The degree to which these parameters varied, including the choice and composition of the porogen, the temperature at which they polymerized, the amount of cross-linking monomer used, and the amount of initiator, affected how well the monolith separated. A reverse-phase HPLC system was used to test the produced monolith for the separation of proteins and oligonucleotides. In reversephase and ion-exchange chromatography, polymer-based monolithic columns are efficient for the high-speed separation of proteins, polypeptides, oligonucleotides, synthetic polymers, and some small molecules, but they exhibit relatively low efficiency for small solutes [51]. They can tolerate extreme changes in pH from 1 to 14 for reversing phase columns. Limitations include their relatively low mechanical stability and compared to conventional polymer beads, organic polymer monoliths provide higher efficiency and less swelling problems. The presence of micropores enhances analyte spreading which causes band broadening and finally leads to large mass transfer kinetics for small molecules [65].

## **5.2 Polar and nonpolar organic monoliths**

Since they are known to have, among other things, the capacity to withstand a wide range of operational conditions and to offer solutions for a variety of separation problems, organic polymer monolithic stationary phases have attracted the attention of the separation science community in the past two decades. In order to adequately describe the numerous parameters involved in the manufacture of polar and nonpolar organic monoliths made of two distinct components, the author of this advanced review paper has attempted to do so. The first section discusses nonpolar organic

monoliths, and the second section discusses polar organic monoliths and variations on them. The methacrylate/acrylate-based monoliths, styrene-divinylbenzene (DVB) based monoliths, and hyper cross-linked monoliths are the three subgroups of the nonpolar organic monolith component. However, the section on polar organic monoliths, which primarily deals with monoliths derived from methacrylates, divides the monoliths into different categories based on the surface-charged groups they contain (for example, neutral, anionic, cationic, and zwitterionic monoliths) as well as how they were functionalized after polymerization [66].

#### *5.2.1 Nonpolar organic monoliths*

There are three commonly used nonpolar organic monolithic stationary phases, namely, acryl amide-based, acrylate/methacrylate-based, and styrene-based monoliths. The latter two are the most suited monoliths for reversed-phase chromatography (RPC) and RP-CEC applications.

#### *5.2.1.1 Acrylate/methacrylate copolymer-based monoliths*

Generally, the acrylate/methacrylate-based monoliths are more polar than their styrene DVB monolith counterparts. The formation of different kinds of monoliths is mainly by the choice of the functional monomers and cross-linking monomers. The structures of the functional and cross-linking monomers used for nonpolar and polar organic monoliths are shown in **Figure 3**.

#### *5.2.1.2 Nature of functional monomers*

The effect of the alkyl chain length and shape of the functional monomer on the structural features of the monolith has been reported. Rathnasekara *et al*. [67] used different alkyl methacrylate monomers with butyl, cyclohexyl,2-ethyl hexyl, lauryl, and stearyl functional groups in the polymerization mixture with ethylene glycol dimethacrylate (EDMA) as the cross-linker. In every instance, 1,4-butanediol (BDO) and 1-propanol were selected as the porogens. Test solutes included conventional proteins and alkylbenzenes, and typical metrics such as column permeability, methylene content, and phenyl selectivity were evaluated. The butyl methacrylate (BMA) monolith had a high level of permeability, whereas the lauryl methacrylate (LMA) monolith had a low level of permeability. Stearyl methacrylate (SMA)-based monolith had the best methylene selectivity, whereas BMA-based monolith displayed the highest phenyl selectivity. By using gradient elution in HPLC, lauryl and cyclohexyl methacrylate offered slightly improved separations for the tested standard proteins. Puangpila *et al*. [68] in CEC also looked at the impact of the functional monomers' alkyl chain length on solute retention. The method was first discussed by Okanda and El Rassi [69] and by Karenga and El Rassi [70]. The authors reported neutral monoliths (empty of fixed charges) to entirely prevent electrostatic interactions formed between charged solutes and the otherwise surface-attached charged moieties.

Using the identical cross-linking monomer pentaerythritol triacrylate, two distinct series of monolithic columns with surface-bound C8, C12, and C16 chains were created (PETA). While a series of monoliths (series B) was prepared by keeping the same composition of functional monomers and cross-linker, it produced chromatographic retention that increased as expected in the order of increasing the n-alkyl chain length. The monoliths (series A) were produced by adjusting the composition of functional

*Monoliths Media: Stationary Phases and Nanoparticles DOI: http://dx.doi.org/10.5772/intechopen.110502*

*Structure of functional monomers and crosslinked used in the preparation of nonpolar and polar monoliths, the monomers and cross-linked arranged alphabetically (Reprinted from Ref. [67]).*

monomers and cross-linker to obtain comparable solute retention regardless of the alkyl chain length. The solvent was a ternary mixture of cyclohexanol, ethylene glycol, and water. The A series' C16 monolith produced the maximum separation performance for tiny solutes, however, the A column series was insufficient for separating proteins. The best separation efficiency for proteins was provided by the C8 monolith of the B series. The C16-monolith of the A series appears to offer the best separation for tryptic peptide mapping. An energetically "harder" C16 surface favored better separation of the smaller-size peptide solutes, whereas an energetically "softer" C8 surface permitted faster sorption kinetics and hence increased efficiency for big protein molecules. Briefly stated, proteins and peptides as well as neutral, polar, and

charged solutes were effectively separated, and the outcomes were consistent with earlier research on neutral monoliths by Karenga and El Rassi [71].

Mixed ligand monolithic (MLM) columns were created for CEC as a revolutionary method of achieving unique selectivity [72]. The creation of these columns involved the copolymerization of various mixtures of the functional monomers octadecyl acrylate (ODA) and 2-naphthyl methacrylate (NAPM) in the presence of the cross-linker trimethylolpropane trimethacrylate (TRIM) and a ternary porogenic solvent made of cyclohexanol, ethylene glycol, and water. In the CEC of neutral, polar, and charged solutes, the combined retentive characteristic of the ODA ligand, which is merely hydrophobic, and that of the NAPM ligand, which is both hydrophobic and an interaction provider, was utilized. As anticipated, the MLM's makeup impacted how large the EOF was. In comparison to the NAPM monomer, the ODA ligand generally showed a higher affinity for the mobile phase ions. This is because the NAPM is a monomer based on methacrylate, whereas the ODA is based on acrylate. **Figure 4** illustrates the discovery that columns formed by either ODA or NAPM alone did not match the unique selectivity for a given set of solutes produced by columns with a specific mix of both ligands.

By co-polymerizing, the functional monomer 3-methylacrylol-3-oxapropyl-3-(N, N-dioctadecylcarbomyl)-propionate (AOD) with the cross-linker EDMA, Duan et al. [73] presented a unique monolithic column with double C-18 chains for RPC. The monolithic column for HPLC was created using fused-silica capillary columns (100 m id). A poly (SMA-co-EDMA) C-18 monolith was also created for the comparison investigations in accordance with the author's earlier work [74]. We employed a binary porogenic combination of BDO and 2-methyl-1-propanol. The back pressure

#### **Figure 4.**

*Electrochromatograms showing the separation of five alkylbenzenes and five PAHs on monolithic columns with different mole fractions ODA/NAPM. Capillary column, 20 cm effective length, 27 cm total length 100 μm id; mobile phase, 70% ACN, 1 mM sodium phosphate monobasic, pH 7.0, running voltage 20 kV; electrokinetic injection for 3 s at 10 kV. Solutes: 1, benzene; 2, toluene, 3,ethylbenzene; 4, 1-nitronaphthalene; 5, butylbenzene; 6, fluorene;7, 9-anthracenecarbonitrile; 8, heptylbenzene; 9, 9-nitroanthracene; and10, fluoranthene. EOF tracer, thiourea (Reprinted from Ref. [72]).*

increased from 0.6 to 13.0 MPa at a flow rate of 500 nL/min as the monomer percentage changed from 50 to 70% w/w. Additionally, it was found that the back pressure increased from 0.6 to 13.0 MPa when the proportion of 2-methyl-1-propanol grew from 75 to 95% w/w. The results obtained were supported by the SEM images. The theoretical plate height of the poly (AOD-co-EDMA) monolith, which was 19.2 mm, was higher than that of the poly (SMA-co-EDMA) monolith, which was 32.1 mm at the same linear velocity of 0.85 mm/s. The permeabilities of this column with ACN, MeOH, and water were good. When strongly acidic and basic buffer were run over a sustained 30-hour period to test chemical stability, no noticeable deterioration was seen. The investigations into the batch-to-batch reproducibility studies and run-to-run repeatability research produced excellent results. In order to assess the effectiveness of the column and the behavior of the RP retention, respectively, Van Deemter plots and methylene selectivity tests were also performed. Using alkylphenols as test solutes, it was found that the methylene selectivity was 1.68, which was comparable to the 1.70obtained using the poly (SMAco-EDMA) monolithic column. Tocopherols (TOH) were utilized to test this column, and as can be shown in **Figure 5**, a complete separation of the, and TOH isomers was achieved in less than 30 minutes.

## *5.2.2 Polar organic monoliths*

Despite offering a variety of chromatographic solutions for the separation, purification, and fractionation of a wide range of solutes, normal-phase chromatography (NPC), which employs polar stationary phases with nonpolar organic mobile phases, has its own limitations in the separation of highly polar analytes. Due to issues with hydrophilic materials dissolving in non-aqueous mobile phases, NPC of hydrophilic samples is challenging [75]. NPC has low repeatability for hydrophilic chemicals and poor mass spectrometry (MS) detection ionization efficiency [76]. Additionally, the

#### **Figure 5.**

*A nano-LC separation of tocopherol homologs on the poly (AOD-co-EDMA) monolithic column. Conditions: Column dimensions 180 mm 100 μm id; mobile phase: MeOH/H2O (93.5/6.5, v/v); detection, 292 nm; flow rate: 500 nL/min; injection volume: 20 nL (Reprinted from Ref. [73]).*

### *Monoliths Media: Stationary Phases and Nanoparticles DOI: http://dx.doi.org/10.5772/intechopen.110502*

majority of hydrophilic samples are poorly maintained on RP columns, eluting close to the column's dead time or displaying little to no resolution.

A chromatographic technique hydrophilic interaction liquid ion chromatography "HILIC" provides an alternative approach to effectively separate small polar compounds on polar stationary phases. In HILIC, polar analytes can be separated more effectively by combining a polar (hydrophilic) stationary phase with an organic-rich hydro-organic mobile phase. The inclusion of organic-rich mobile phase in HILIC offers some additional benefits that will increase the utilization of this method. These benefits include HILIC's appropriateness for direct coupling with MS detection and low column back pressure, which facilitates quick analyte separation with shorter analysis times [77]. The retention of polar solutes in HILIC and hydrophilic interaction CEC is strongly influenced by the makeup of the mobile phase (HI-CEC). HILIC/HI-CEC is typically operated with a low aqueous, highly organic mobile phase. The monolithic surface develops a water-rich layer under these HILIC circumstances. The adsorbed water layer on the surface of the stationary phase, the hydro-organic mobile phase, and the polar solutes in between are partitioned to create the separation. While HILIC frequently offers an efficient separation for polar analytes, a straightforward retention mechanism is not achievable for most molecules because hydrogen bonding, dipole–dipole, ion-dipole, and ion-ion interactions are also involved in addition to a partition mechanism [77].

During the past two decades, porous organic polymer monolithic columns have been widely employed as HILIC stationary phases due to their particular benefits, which include (i) high permeability, which reduces backpressure, (ii) low resistance to mass transfer, (iii) easy fabrication and surface functionalization, (iv) stability under severe pH values, and (v) a variety of functional monomers [78]. A variety of organic polymer monoliths with amino [79, 80], amide [81], hydroxyl [81], sulfoalkylbetaine [82], boronic acid [83], anilines and benzoic acids [84], and some other functionalities have been reported for use in HILIC/HI-CEC separations. **Table 1** summarizes the polar monoliths (neutral-cationic-anionic-zwitterionic monolith) and



#### **Table 1.**

*Summary of polar monolith (neutral-cationic-anionic-zwitterionic monolith) and their applications for the separation of analytes.*

their applications for the separation of analytes. Applications using polar organic monolith are shown in **Figures 6** and **7**.

#### **5.3 Monolithic metal: organic framework**

#### *5.3.1 A sol: Gel monolithic metal: Organic framework*

The creation of a porous monolithic metal–organic framework (MOF) with a 259 cm3 (STP) capacity following effective packing and densification. This is the greatest value for conforming shape porous solids documented to date, and it represents a more than 50% improvement above any experimental value previously reported. A significant advancement has been made in the use of mechanically robust conformed and densified MOFs for high volumetric energy storage and other industrial applications when nanoindentation tests on the monolithic MOF revealed robust mechanical properties, with hardness at least 130% greater than that previously measured in its conventional MOF counterparts [85].

#### **Figure 6.**

*Electrochromatographic profiling of phenols (A) and nucleic acid bases and nucleosides (B) on poly(META-co-PETA monolith. The profile (A) was recorded in the mobile phase 5 mM ammonium formate, pH 3.0, at 95% v/v ACN; pump flow: 0.02 mL/min; backpressure 250 psi; applied voltage: +10 kV. Solutes: (1) phenol; (2) catechol; (3) hydroquinone; (4) resorcinol; (5) pyrogallol. The profile (B) was recorded in the mobile phase 5 mM ammonium formate, pH 5.0, at 90% v/v ACN; the other conditions were the same as for (A). Solutes: (1) uracil; (2) adenine; (3) adenosine; (4) cytosine; (5) uridine; (6)guanine; (7) cytidine; (8) guanosine (Reprinted from Ref. [79]).*

#### **Figure 7.**

*Separation of three phenols (A), four phenol derivatives (B), and three other compounds (C) obtained on the neutral hydroxymonolith. Condition: Hydro-organic mobile phase, 5 mM NH4Ac (pH 8.0) at 95% ACN (v/v), running voltage 20 kV, column temperature 20°C, sample injection, pressure at 5 bar for 10 s. solutes in C are DMF, formamide, and thiourea (left to right) (Reprinted from Ref. [81]).*

#### *5.3.2 Metal–organic frameworks in biomedicine*

#### *5.3.2.1 Influence of the composition*

For biomedical applications, porous materials must have a composition that is conducive to living things. There are not many toxicity studies that involve MOFs or coordination polymers at this time. Most of the information is limited to the assessment of the individual toxicity of the metals and linkers. Of course, only metals with a tolerable level of toxicity should be taken into account here. The choice to exclude a certain composition from biomedical use could be based on a number of factors, including the application, the trade-off between risk and benefit, and the kinetics of degradation, biodistribution, accumulation in tissues and organs, and excretion from the body. All metals and linkers could therefore be employed in these applications, although at varying doses based on the aforementioned requirements. According to their oral lethal dosage 50 (LD50), the most suitable metals at first look are Ca, Mg, Zn, Fe, Ti, or Zr, whose toxicity ranges from a few g/kg to more than 1 g/kg (calcium). The most popular one is the use of exogenous linkers, either made synthetically or naturally from substances that do not interfere with bodily cycles. Relevant exogenous MOFs for bio-applications include those made from iron (III) polycarboxylates, such as MIL-100(Fe) [86], zinc adeninate-4,40 biphenyldicarboxylate BioMOF-1 [87, 88], and magnesium coordination polymers, such as the magnesium 2,5-dihydroxoterephthalate CPO-27(Mg) (CPO for Coordination Polymer from Oslo) [89] (**Figure 8**).

#### **Figure 8.**

*View of the structures of a few topical MOFs, here CPO-27(Mg, Zn) (left), MIL-100 (Fe) (center), and Bio-MOF-11 (right), based on exogenous linkers for bioapplications. Metal polyhedra and carbon atoms are in blue (Zn, Mg) or orange (Fe), and black, respectively (Reprinted from Ref. [88]).*

These solids have large pores (4–29) and surfaces between 1200 and 2200 m<sup>2</sup> . Biomolecules (NO, CO, H2S, medicines, etc.) may coordinate strongly at these accessible Lewis acid sites to better control the release [90–92]. The structures of a few MOFs based on endogenous linkers such as Bi citrate [93], Mg formate [94], Cu aspartate [95], Zn-dipeptide [96], Fe gallate [97], Fe Fumarate [98] and Fe muconate [99] are shown in **Figure 9**.

#### **Figure 9.**

*View of the structures of a few MOFs based on endogenous linkers such as; Bi citrate [93], Mg formate [94], Cu aspartate [95], Zn-dipeptide [96], Fe gallate [97], Fe fumarate [98], and Fe muconate [99]. Metal polyhedral are in pink, gray, gray, blue, or orange (for Bi, Mg, Zn, Cu, and Fe, respectively) and carbon atoms in black, respectively.*

Exogenous linkers should be eliminated from the body following the *in vivo* delivery of the MOFs in order to prevent potentially harmful side effects. As a result of their high polarity and ease of removal under physiological conditions, typical polycarboxylic or imidazolate linkers are not initially thought to be very toxic, with rat oral doses of 1.13, 5.5, and 8.4 g/kg for terephthalic, trimesic, 2,6-napthalenedicarboxylic acid, and 1-methylimidazole, respectively.

The use of functionalized linkers to adjust MOFs' absorption, distribution, metabolism, and excretion is yet another potential application for exogenous linker-based MOFs (ADME). Additionally, the presence of functional groups inside the framework can alter the host-guest interactions for the adsorption and distribution of therapeutic molecules, enabling a better control of the release. While a number of porous MOFs based on modified linkers that contain polar or apolar functional groups such as amino, nitro, chloro, bromo, carboxylate, methyl, and perfluoro are known, the most prevalent functionalized systems involve porous metal terephthalates based on iron or zinc [100]. One could also cite a series of organically modified porous Zn imidazolate solids [101]. In the case of functions, flexible MOFs will not only modify the host and guest interactions, but will also drastically affect the flexibility of the MOF during the adsorption or delivery of the biomolecule [102, 103] (**Figure 10**).

## *5.3.2.2 Nanoparticle monoliths*

For some administration routes, where highly exact sizes are needed, particle size is a limiting constraint. For instance, the parenteral method necessitates stable

**Figure 10.** *Scheme of biodistribution of iron nanoMOFs according with the iron concentration (Reprinted from Ref. [88]).* solutions or suspensions of nanoparticles smaller than 200 nm in order for them to freely flow through the tiniest capillaries. Therefore, the creation of homogenous, monodispersed, and stable nanoparticles is a significant issue that has been addressed thus far using the following techniques:

### *5.3.2.3 The conventional hydro/solvothermal route*

The conventional hydro/solvothermal method depends on a number of parameters, including reaction duration, temperature, stoichiometry, dilution pH, additives, etc. Topical examples include the porous zinc terephthalate MOF-5 (100–200 nm) [104] or the flexible porous Iron (III) dicarboxylates MIL-88A (150 nm) [105], MIL-88B-4CH3 (40 nm) [105], obtained by reducing reaction time or temperature, either employing low temperature or atmospheric pressure conditions. Alcohols can also be used to create nanoparticles of the zinc imidazolate ZIF-8 (40 nm) or the porous iron muconate MIL-89 (30 nm) [106] at low temperatures. Although particle sizes less than 100 nm are frequently achieved, the lack of homogenous and efficient heating typically results in a significant drop in yield and a high degree of polydispersity because the nucleation and growth stages are not under control [107]. Acidobasic or inhibitory additives (acetic acid, hydroxybenzoic acid, and pyridine) [106] are frequently used to adjust the reaction kinetics or slow the nucleation development process. Pyridine has been suggested as an inhibitor in the solvothermal production of porous indium terephthalate particles by Cho *et al*. [108]. Acetate ions were utilized by Horcajada *et al*. [106] to create tiny nanoparticles of the malleable, porous iron muconate MIL-89 as growth inhibitors (30 nm). Similar results were obtained by Tsuruoka et al. [109] with porous Cu2-(naphthalene dicarboxylate) 2(1,4 diazabicyclo (2,2,2) octane nanorods. A polyvinylpyrrolidone (PVP) polymer was used by Kerbellec *et al*. [110] to produce extremely tiny nanoparticles of a luminous terbium terephthalate (about 4–5 nm) at ambient pressure and room temperature. This approach was additionally used for various lanthanide (Tb, La, Tm, or Y) terephthalate MOF luminous micro- and nanoparticles [111].

#### *5.3.2.4 Reverse-phase microemulsions*

Reverse-phase microemulsions are based on a metal source, an organic linker, and micelles of cationic cetyltrimethylammonium bromide surfactant (CTAB) in a nonporous Ln [112–114] or Mn [115] based polycarboxylates MOFs with interesting imaging properties. This technique allows a control of particle size by tuning the dimensions of the micelles. Isooctane/1-hexanol/water mixture led to nonmetric nonporous Ln [112–114] or Mn [115] based polycarboxylates MOFs with interesting imaging properties technique allows a control of particle size by tuning the dimensions of the micelles.

### *5.3.2.5 Sonochemical synthesis*

Micro and nanoscale MOFs have recently been synthesized using the quick, simple, and ecologically friendly sonochemical synthesis technique. Acoustic cavitation is caused by ultrasonic irradiation and results in the bursting of bubbles, localized hot patches, a significant temperature/pressure differential, and quick molecular movement. This encourages the development of high-energy microreactors, which causes MOFs to crystallize quickly [116, 117]. While other porous crystalline structures have

been successfully created at the nanometric scale under ultrasonic circumstances, the microporous flexible iron(III) terephthalate MIL-5376 and a stiff copper trimesate have been crystallized at the microscopic scale [118, 119]. The Seo research group has detailed the ultrasonic synthesis of porous copper trimesateHKUST-1 in DMF (10– 200 nm) and zinc trimesate in ethanol (50–100 nm) at room temperature and ambient pressure using selective organoamine sensing [120]. The flexible porous iron fumarate MIL-88A's particle size can be varied between 100 and 740 nm, with the lowest particle sizes being obtained by using inhibitors, extremely high dilutions (0.01–0.008 M), or low temperatures (°C). As a result, only low yields (5 wt%) are produced [107].

#### *5.3.2.6 Microwave-assisted hydro/solvothermal synthesis*

For the creation of MOF nanoparticles, microwave-assisted hydro/solvothermal synthesis is an effective, homogeneous, and quick technique. The high thermal efficiency of polar solvents results in local superheated regions and high dielectric absorptivity, which favors a rapid and homogeneous nucleation process over crystal formation [121]. This technique was used to produce the zinc terephthalates IRMOF-1, 2, and 3 (100 nm) [122] and the mesoporous chromium terephthalateMIL-101 (22 nm) [123, 124]. Sefcjk and McCormick [125] have extensively studied the flexible microporous iron terephthalate MIL-53 (350–1000 nm), the mesoporous iron trimesate MIL-100 (200 nm), the iron aminoterephthalate MIL-101NH2 (120 nm) and the iron fumarate MIL-88A (20 nm), as shown in **Figure 8**.

#### **5.4 Silica monoliths**

Sol–gel synthesis, which enables extraordinary control over the composition and shape of the generated monolith, is typically used to create silicon alkoxide-based monoliths. This method is flexible and may be used to create a variety of monoliths. Starting with Si(OR)4, the reaction creates a siloxane (Si-O-Si) network in the polymer. Phase separation, condensation, and hydrolysis compete with one another to create a porous monolith [125]. There were several steps involved in creating the silicon alkoxide-based monolith. Alkoxy groups are replaced by hydroxyl groups in the first step of the process, which also results in the synthesis of silanol groups (SiOH) and alcohol. These extremely reactive silanol groups condense with additional alkoxy silanes or with one another in the next steps, creating a siloxane bond. Following these preparatory phases, a soil's precursors hydrolyze under acidic or basic conditions, forming a gel with a three-dimensional network following a series of condensation steps. To create the monolithic matrix, this gel ages and passes through phase separation under a certain set of circumstances [125]. Tetramethoxysilane (TMOS) and tetraethoxysilane (TEOS) are typically the two precursors that are employed the most in the preparation. By regulating the rate of condensation and hydrolysis, the reactivity of the precursors can regulate the distribution of subunits throughout the network. Several species are produced as a result of the reaction, and they all proceed through hydrolytic polycondensation at different speeds than the precursor. When compared to other precursors, TMOS undergoes rapid hydrolysis, making it one of the most reactive [126]. When compared to TEOS, it was found that TMOS produces pores that are more consistent, thinner, and have a higher surface area. Various strategies have been developed to address the issues of cracking and shrinkage. Despite various concessions in their performance or stationary phase

*Monoliths Media: Stationary Phases and Nanoparticles DOI: http://dx.doi.org/10.5772/intechopen.110502*

qualities, they proved to be useful. The initial method that distinguished the development was the creation of particle-loaded monoliths in a capillary format with the goal of creating a column without cracks. This method of preparation was first presented by Dulay *et al*. [127] with the idea of embedding particles inside the holes or cavities made inside the produced matrix. Using sol–gel technique, monoliths were created by embedding ODS particles (3–5 m) in tetraethoxysilane (TEOS). A 40 cm long, 75 m capillary was filled with the solution, and the resulting monolith was employed in CEC. A combination of aromatic and non-aromatic chemicals was used to assess the column's performance. Due to the ODS particles' deep embedding and nonhomogeneous packing, which shields the particles from the analyst, different efficiency is attributed to these factors. Bakry *et al*. [128] adapted the above technique and enclosed silica particles within the polymeric backbone to address these issues (polystyrene divinylbenzene). In their process, silica particles were first packed into prepared silica capillary using the slurry packing method, and then an immobilizing fluid made up of styrene, divinyl benzene, azobisisobutyronitrile (AIBN), and decanol was added. After polymerization, the created monolith underwent a test to determine whether polyphenols, peptides, and proteins could be separated. Sometimes it is simpler to synthesize monoliths made in capillaries than bigger diameter columns because they can form a covalent bond with the inner capillary wall, which gives the monolithic bed more stability.

#### **5.5 Hybrid monoliths**

The sol–gel method, which entails a succession of hydrolysis and polycondensation stages to generate a three-dimensional network structure, is also used to prepare organic–inorganic hybrid monolithic materials [129]. Except for the copolymerization of a second precursor, which is in charge of adding an organic moiety to the silica backbone to prepare a desirable stationary phase, the preparation techniques are the same as in the single alkoxide production. The addition of one or more modified precursors, such as 3-mercaptopropyltrimethoxysilane (MPTS) [130], sets it apart from pure organic and inorganic-based monoliths. The organic moiety is uniformly distributed throughout the matrix and connected to the expanding silicon network *via* a non-hydrolyzable Si-C bond, which improves the performance and efficiency of the monolith to the desired chromatography. Allyl, octadecyl, phenyl, and amino groups were adsorbed onto the matrix, eliminating the need for the timeconsuming and laborious post-matrix modification issue [131–133].

As a replacement for the current stationary phases, a hybrid monolith made utilizing a sol–gel process and hybrid materials have been proposed. This hybrid monolith has shown promise because it offers more benefits than a traditional silica monolith. In order to increase column efficiency, stability, and selectivity, sol–gel hybrid materials are specifically created to have desired qualities and eliminate the ones that are undesired. Additionally, hybrid monoliths can be created directly by combining organic and inorganic monomers for the desired interaction, negating the need for the functionalization of stationary phases, which is more typical with the conventional method, which involves creating the monolith first and then functionalizing it. Therefore, Hayes and Malik [134] created the monoliths using the solution without using particles and without the necessity for frits in their hunt for a substitute to combine the preparation and functionalization of silica monolith in a single step. They provided a straightforward method for creating functionalized porous monoliths that are chemically adhered to the silica capillary's inner walls. The sol–gel precursor,

N-octadecyldimethyl [3-(trimethoxysilyl) propyl] ammonium chloride, was employed to create porous monoliths helpful for CEC studies with chemically bound ODS ligands. Later, employing methyltrimethoxy silane (MTMS) alone as a precursor, Laschober *et al*. [135] developed a capillary monolith based on the sol–gel process, which has the advantage of having increased hydrolytic stability of the Si-C bond. Additionally, they looked at how different parameters affected the monolith's morphology, which was shown by the size of its pores, skeleton, and surface. The prepared monolith is usable throughout a wider pH range. Due to their decreased compatibility with polar solvents, the monoliths produced by MTMS showed a higher tendency for spinoidal decomposition than tetraalkoxysilanes. Furthermore, unlike the case with tetraalkoxysilanes, synthesis does not call for the functionalization of the capillary walls or a hydrophilic polymer like polyethylene glycol. At pH 1, the reaction is conducted to create monoliths with bicontinuous morphologies. When compared to tetralkoxysilane, the surface area is less, which may be due to the involvement of only three of silicon's four valences in forming bonds with other organo-silica tetrahedrons. The monolith's chromatographic performance was evaluated using each component separately, and separation was predicted based on the components' various retention times. Dunn and Zink [136] recently evaluated the characteristics and uses of molecules trapped in a silica matrix made using the sol–gel method. In order to explore the impacts of changes in many factors, such as solvent composition, polarity, viscosity, pH, and speeds of chemical reaction, entrapped molecules acting as a spectroscopic probe offer insight into sol–gel chemistry. As probes, a variety of organic compounds have been used to track the chemical changes in sol–gels.

## **6. Preparation of hydrophilic monolithic columns**

## **6.1 Silica-based monolithic columns**

The Nakanishi and Soga team invented silica-based monolithic columns in the early 1990s [137]. The majority of these silica-based monoliths that have been described were created using methods that are comparable and involve the hydrolysis of one or more silanes, primarily tetramethoxysilane, in an acidic solution while the presence of an appropriate porogen. A sufficient mesopore is formed when the gel is aged and macerated in a basic solution. The gel is then dried and heated before having its surface modified with the appropriate ligands. One particular benefit of the silicabased monolithic column is that its macropores and mesopores can be tailored separately to achieve the best performance for specific analytical objectives. This is in addition to the two main generic advantages of all monolithic columns, namely, the high permeability and the low resistance to mass transfer. According to Jiang *et al*. [138], the majority of articles on silica monolithic columns dealt with applications based on commercial columns. He hypothesized that this was due to the fact that several stages (such as drying and cladding of the rods) are challenging to overcome in academic laboratories and that Merck, the producer, has carefully crafted patents designed to close any gaps [139]. The Chromolith Performance monolith, one of Merck's commercially available silica monolithic columns, has the potential for HILIC applications because it features a polar silica surface. However, based on these Chromolith Performance materials, just one such HILIC application has been documented. This column was employed by Pack and Risley [140] for the detection and quantification of lithium, sodium, and potassium in the HILIC separation mode.

*Monoliths Media: Stationary Phases and Nanoparticles DOI: http://dx.doi.org/10.5772/intechopen.110502*

Additionally, certain monolithic capillary columns made entirely of silica have been created in academic labs and used for HILIC. The first silica-based monolithic capillary columns were created at the end of the 1990s [137] using the sol–gel method described by Nakanishi and Soga. However, the drying and calcination processes involved in its preparation take time. Additionally, Puy *et al*. [141], a 2 h water washing phase, which may remove 70% of the polyethylene glycol, was used in place of the 24 h calcination step (PEG). The monolith's stability, morphology, or chromatographic qualities are unaffected by the residual PEG. Later, they discovered that monolithic capillaries made of pure silica and employed in HILIC mode or normal phase mode did not require the hydrothermal treatment step at 120°C. The hydrothermal treatment was suppressed, which increased the retention factors but did not affect CEC or nano-LC efficiency [142]. The monolithic silica matrix synthesized from a sol–gel process was chemically modified by 3-aminopropyltrimethoxysilane, 3-(2 aminoethylamino) propyltrimethoxysilane [138], or diethylenetriaminopropyltrimethoxy silane [143] in order to produce a column for hydrophilic interaction applications. The surface modifications were simply carried out by a solution of

pumping the silane in anhydrous toluene through the bare silica monolithic capillary column for 1 h which was thermostated at 75°C or 110°C for various times. These amino silica monolithic stationary phases exhibited HILIC behavior toward neutral solutes. However, no comparative research between these columns and bare silica monoliths has been reported. Moreover, Ikegami *et al.* [144] summarized a polymer coating procedure in their review article as shown in **Figure 11**.

#### *6.1.1 Functionalization of silica-based monoliths*

The study on the modification or functionalization of silica-based monolithic columns has been developed to satisfy the varied needs for the separation of complex substances in LC and CEC based on the silica-based monolithic columns created utilizing the aforementioned procedures. **Figure 12** illustrates how monolithic columns can entirely independently manage the chemical and porous properties by postmodification. Additionally, post-modification allows for the avoidance of the need to prepare a monolithic column's porous characteristics from scratch each time a change in chemical functionality is sought. For the functionalization of porous silica-based monoliths, a number of approaches have been identified. The far simpler method to change the silica-based monolithic columns, as shown in **Figure 13** [127], was to use various silane chemicals.

## **7. Selected monoliths media applications**

Applications developed for monoliths media have been tremendous, and it would be impossible to review them all in this chapter book. We have, however, categorize studies of monoliths media with metal–organic framework and nanoparticle monoliths in chromatography separations. Some representative examples published over the last decade are summarized below to provide the reader with an appreciation of the possible breadth of applications.

**Figure 12.** *Post-modification of silica monolith (Reprinted from Ref. [144]).*

*Monoliths Media: Stationary Phases and Nanoparticles DOI: http://dx.doi.org/10.5772/intechopen.110502*

**Figure 13.** *List of silane reagents for silica-based monolithic stationary phases (Reprinted from Ref. [127]).*

## **7.1 Applications of MOFs in LC**

The use of MOFs for the LC separation of compounds, such as various aromatic molecules [145–147], medications [148], dyes [149, 150], pollutants [151], and peptides [152], has attracted a lot of interest. In fact, the early MOF-based HPLC separation of xylene isomers and ethylbenzene employed MIL-47 as the stationary phase. It was then employed to extract styrene and ethylbenzene [153]. Other MOFs that have been proposed as HPLC stationary phases include ZIF-8 [154], MIL-53 [155, 156], MIL-101 [157], MIL-47 [158], and MIL-53 [155, 156]. Direct packing of MOF particles, on the other hand, led to low separation efficiency, undesired peak morphologies, and large column backpressures due to their irregular forms, sub-micrometer size, and wide size ranges. It can be challenging to regulate the pore size of silica deposition substrates and the synthesis of MOFs [159]. One method utilized to separate xylene isomers, dichlorobenzene, and chlorotoluene isomers, as well as ethylbenzene and styrene, was a MIL-101(Cr) packed column (5 cm 4.6 mm i.d.) [157]. For ethylbenzene, the packed column's efficiency was just 20,000 plates per meter, which is not particularly noteworthy. In comparison to the meta- and para-isomers, the affinities for o-dichlorobenzene and o-chlorotoluene were greater. These early MOF publications frequently omitted the fact that alternative, frequently commercial, LC columns had been able to separate these compounds more effectively for years.

The deposition of MOF thin films onto core-shell silica particles was regulated using a layer-by-layer deposition technique [160]. In this work, MOFs such MIL-101 (Fe) NH2 (Fe3(O)(BDC NH2)3(OH)(H2O)2, BDC = benzene-1,4-dicarboxylic acid, and UiO-67 (Zr6O4(OH)4(BPDC)6, BPDC = 4,4<sup>0</sup> -biphenyldicarboxylate) were employed. Mixed-mode liquid chromatography was used to manufacture and analyze metal–organic framework-assisted hydrogel-bonded silica composite microspheres (Zn-BTC MOF, DNs-hydrogel@SiO2) [161]. A more tolerable efficiency for this kind of chromatographic column is 90,300 plates per meter (sucrose). Alkylbenzenes, organic acids, carbohydrates, a few antibiotics, polycyclic aromatic hydrocarbons, insecticides, and anions could all be isolated from nucleosides and their bases (**Figure 14**). All of them have, of course, been readily separable on a variety of commercial stationary phases for many years. A chiral sorbent made of a Zn + 2-based MOF with L-lactic acid ligands was utilized to extract a variety of sulfoxide enantiomers [162]. Additionally, it was utilized in chiral alkyl aryl sulfoxides' traditional LC separation. In open tubular capillary electrochromatography, a different D-(+) camphoric acid + Zn + 2-based MOF was utilized to separate the enantiomers of flavanone and praziquantel [163]. Additionally, several achiral compounds were isolated. Magnetic particles were utilized in conjunction with another chiral MOF to

#### **Figure 14.**

*Chromatograms for the separation of (a) alkylbenzenes, (b) polycyclic aromatic hydrocarbons, and (c) pesticides on DNs-hydrogel@SiO2 column. Analytes: (a) 1. Benzene, 2. Methylbenzene, 3. Ethylbenzene, 4. Propylbenzene. 5, butylbenzene. 6, Pentylbenzene; (b) 1. Benzene, 2. Naphthalene, 3.2-methylnaphthalene, 4, fluorene, 5. Phenanthrene, 6. Fluoranthene, 7. Benzoanthracene; (c) 1. Flufenoxuron, 2. Meturon, 3. Chlortoluron, 4. Diuron, 5. Diflubenzuron. Mobile phase: (a) ACN/H2O (40/60, v/v); (b) ACN/H2O (38/62, v/v) and (c) ACN/H2O (35/65, v/v). UV detection at 254 nm. Column temperature: 25°C. flow rate: 1.0 mLmin-1 (Reprinted from Ref. [161]).*

#### **Figure 15.**

*Representative chromatograms for the separation of* trans*-2,3-diphenyloxirane enantiomers with HPLC column packed with TAMOF-1: (a) separation with different mobile phases using column A: Isopropanol, methanol, acetonitrile, and 95:5 hexanes/isopropanol (v/v). (b) Comparison with commercial columns CHIRALPAK®- AD, CHIRALCEL®-OD and CHIRALCEL®-OJ in 95:5 hexanes/isopropanol (v/v) (Reprinted from Ref. [165]).*

enantioselectively enrich an enantiomeric pharmacological intermediate from the solution [164].

It has been claimed that the porous and durable homochiral (MOF) TAMOF-1, which was made from copper (II) and a natural L-histidine linker, can separate a few racemic combinations, including several medications [165]. There have been claims that it performs better than some commercial HPLC chiral columns, but the outcomes do not appear to back up even this extremely superficial comparison (**Figure 15**). For the enantioseparation of 18 racemates, including alcohols, phenols, amines, ketones, and organic acids, a similar homochiral D-his-ZIF-8@SiO2 composite was tested [166]. The separations were carried out using n-hexane/isopropanol as the solvent in the standard phase mode. For reversed phase separations in capillary electrochromatography, a monolithic column made of 1-allyl-methylimidazolium chloride (AlMeIm + Cl-) copolymerized with ZIF-8 was utilized [167]. This method produced monolithic columns that were used to separate neutral chemicals, anilines, and phenols. The performance of the column efficiency was improved by the synergistic interaction between the ionic liquid and ZIF-8. The maximum column efficiency was found in toluene, which measured 2.07<sup>10</sup><sup>5</sup> theoretical plates m<sup>1</sup> .

### **7.2 Applications of nanoparticle-based monoliths**

#### *7.2.1 Gold nanoparticles*

Due to their simple and inexpensive synthesis, huge surface area, molecular recognition properties, and compatibility with living things, gold nanoparticles (GNPs) are among the nanomaterials that have been the subject of the most research. GNPs have been used in a variety of contexts and are receiving more interest in the monolithic sphere. A new silica monolithic stationary phase functionalized with octadecanethiol GNPs for CEC was created by Ye *et al*. [168]. In response to neutral solutes, the resulting GNP-modified silica monolith displayed normal reversed-phase

electrochromatographic activity. Due to the great affinity of gold for these moieties, GNPs can be covalently linked to surfaces containing amino, thiol, or cyano functionalities [169]. A-glucosidase was then easily and stably immobilized onto GNPs due to the strong affinity between gold and enzyme amino groups, which was achieved by the same group [170] by covalently attaching GNPs to the surface of a polymer monolith *via* the creation of an Au-S bond. Additionally, silica monoliths coated with bovine serum albumin-gold nanoparticles (BSA-GNPs) conjugate were reported to be employed as chiral stationary phases for the CEC enantioseparation of various phenylthiocarbamyl amino acids [171]. Lv and colleagues [172] created a novel method for creating porous polymer monoliths with improved GNP coverage of pore surfaces. This method considerably improved the immobilization of GNPs, and it was discovered that the density of pore surface covering was significantly influenced by the size of the GNPs. As a "universal" intermediate ligand, the surface of the attached gold was subsequently functionalized with 1-octanethiol and 1-octadecanethiol to produce the desired monoliths for the separation of proteins in reversed-phase mode. In **Figure 16**, three proteins are separated in a gradient of acetonitrile (ACN), with the order of elution determined by the hydrophobicity of the proteins. Monoliths modified with 15, 20, and 30 nm NPs provided the best separations because these sizes produced the densest covering of the pore surface with GNPs. The combination of GNPs attached by layered architecture to hypercrosslinked polymer-based monoliths and functionalized with hydrophilic properties was initially described by the same group [173]. With its use in the separation of tiny polar analytes, such as nucleosides and peptides, under hydrophilic interaction chromatographic conditions, this effective monolithic stationary phase was proven.

#### *7.2.2 Silver nanoparticles*

The advantages of silver nanoparticles (AgNPs) over other nanostructured metal particles, such as their advantageous electrical conductivities [174], antibacterial activities [175], and optical properties [176], are well established. Monolithic columns with embedded AgNPs were created and tested in order to combine the unique properties of AgNPs with monoliths [177]. Investigated how the presence of AgNPs affected the polymer matrix's morphological and chromatographic characteristics. AgNPs have a very strong affinity for the anions of heavy halogenides like astatide and iodide. In order to remove these constituents from solutions, AgNPs may, therefore, be a great candidate [178]. The creation of a macroporous monolithic column with anchored AgNPs for the removal of extra radioiodine from a radiolabeled pharmaceutical was demonstrated by Sedlacek et al. [179].

## *7.2.3 SiO2/TiO2 nanoparticles*

As a result of their better chemical affinities and amphoteric characteristics, metal oxides have been introduced into monolithic structures with increasing effort. Although TiO2 has received the greatest attention and is the most often utilized metal oxide, few research studies have successfully created monolithic capillary columns made entirely of pure TiO2 [180, 181]. Without a doubt, creating monoliths based on TiO2 presents a number of difficulties. Additionally, according to several related reports, SiO2/TiO2 composite materials outperform pure TiO2 materials in the enrichment of phosphorylated targets [182–184]. Wang *et al.* [185] developed a facile sol–gel method for preparing a novel SiO2/TiO2 in-capillary composite monolith under mild

*Monoliths Media: Stationary Phases and Nanoparticles DOI: http://dx.doi.org/10.5772/intechopen.110502*

#### **Figure 16.**

*Reversed-phase separation of proteins using monolithic columns containing GNPs modified with 1-octanethiol. Conditions: Columns: (a) 5 nm GNP, 100 mm 100 mm I.D.; (b) 10 nm GNP, 110 mm \_ 100 mm I.D.; (c) 15 nm GNP, 110 mm 100 mm I.D.; (d) 20 nm GNP, 108 mm 100 mm I.D.; (e) 30 nm GNP, 80 mm 100 mm I.D.; and (f) 40 nm GNP, 89 mm 100 mm I.D.; mobile phase: A 0.1% aqueous trifluoroacetic acid, B 0.1% trifluoroacetic acid in ACN, gradient from 20 to 70% B in A in 20 min, flow rate: 1.0 ml min<sup>1</sup> , injection volume: 10 nl, detection wavelength: 210 nm. Peaks: (1) impurity from ribonuclease A, (2) ribonuclease A, (3) cytochrome C, and (4) myoglobin (Reprinted from Ref. [172]).*

conditions, which was successfully applied to specifically capture phosphopeptides as a metal–oxide affinity chromatography material.

#### *7.2.4 Zirconia nanoparticles*

Zirconia is a desirable and appropriate substitute for silica as the support, primarily due to its exceptional and one-of-a-kind chemical, mechanical, and thermal stabilities [186–188]. The benefits of zirconia NPs include the fact that they are stable at temperatures up to 200°C and exhibit no discernible evidence of disintegration over a wide pH range. Zirconia-based stationary phases can avoid certain notable limitations of silica-based stationary phases, such as restricted use in a constrained pH range and temperature range. Zirconia's distinct surface chemistry, meanwhile, expands the range of chromatographic separation applications [189]. A porous zirconia monolith (ZM) modified with cellulose 3,5-dimethylphenylcarbamate (CDMPC) was created by Kumar and Park [190] and employed as a chiral stationary phase for the CEC separation of a group of fundamental chiral chemicals.

Subsequently, they reported other zirconia-based chiral stationary phases for the CEC separation of β-blockers [191], chiral acids and bases [192], basic compounds [193], and other chiral analytes [194].

#### *7.2.5 Silica nanoparticles*

Liu *et al.* [195] developed a novel cage-like silica NP-functionalized silica hybrid monolith based on the "one-pot" method using thiolene click chemistry. The resulting hybrid monolithic column was characterized and evaluated, and the results showed that it possessed homogeneous macroporous morphology, high permeability, and a strong EOF throughout a wide pH range from 2.7 to 11.2. On the constructed monolith, anilines and phenols were effectively separated by CEC, and 2-aminophenol demonstrated the best theoretical efficiency of 470,000 N m<sup>1</sup> . Reversed-phase and cation-exchange interactions served as the basic foundation for the retention mechanisms. In order to create organic-silica hybrid monoliths, this work illustrated a novel technique for directly integrating modified nanoparticle monomers.

## **8. Challenges and future trends**

There is still a need for additional work to design distinct inventive NP-based monoliths for various particular applications. The majority of pertinent studies in this sector are, however, still in their infancy, and study in this area has only lately begun, thus there are still many restrictions and difficulties. The issue with characterization is the first worry. It will take further work to fully characterize the substrate, including its chemistry, functional surface, and stability. Because most separation mechanisms are still not fully known, a further obstacle lies in the extensive research in theoretical studies. Furthermore, there is still significant work to be done to ensure their reliable and repeatable production in actual applications, as opposed to only for standard separations. Studies on NPs are still ongoing, with a focus on how they can be used in monolithic matrices for chromatographic separations. These studies aim to control the selectivity of the separations as well as the overall performance of the chromatography by designing and synthesizing highly selective interaction sites on the surfaces of NPs and functionalizing monolithic matrices with a wider variety of NPs bearing different functional groups. In-depth research should also be done on the creation of new functionalization techniques that enable the encapsulation of a greater number of NPs in the polymerization mixture. Additionally, it is important to note that research on innovative organic–inorganic hybrid monolithic columns containing NPs is still in its infancy. Instead, the majority of recent investigations have mostly focused on porous polymer monolithic stationary phase as the matrix for functionalization with NPs. Further research into these NP-modified hybrid materials is therefore necessary in order to uncover novel solutions to some long-standing issues and provide fresh ideas for expanding the extensive application of NPs in chromatography.

*Monoliths Media: Stationary Phases and Nanoparticles DOI: http://dx.doi.org/10.5772/intechopen.110502*

Significant advancements have also been made in the functional applications, controlled synthesis, and structural design of MOFs. However, their lackluster stability and exorbitant price restrict their usefulness. MOFs with porous or nonporous silicas can be combined to improve stability and characteristics, and the resulting composites can be used for a variety of applications. Another obstacle is the lack of commercially accessible MOF-based materials for dynamic LC separations, but given how quickly this subject is developing and how many studies have been done in this area, this may not be too far off from becoming a reality. Industry demands that stationary phases be stable in the presence of both water and organic solvents. Additionally, a greater study is required on MOF properties and the variables affecting them. Future research should concentrate on improving existing models that are applicable to MOF synthesis circumstances and creating computer tools that will help researchers improve the conditions and take other elements into account.

## **9. Concluding remarks**

Due to the distinctive qualities of the porous monolithic method, such as simple production, superior permeability, and quick mass transfer, monolithic matrices have attracted increasing interest in the field of liquid chromatographic separation. Monolithic stationary phases have been extensively used in hyphenated systems coupled with mass spectrometers, miniaturized devices, and quick and high-efficiency oneand multi-dimensional separation systems. The separation of complex biological materials using columns is being revolutionized by new monolithic stationary phase preparation technologies. Direct synthesis is a convenient and versatile route to prepare the monolithic columns for microscale separation. Whereas obtaining desired monoliths with chromatographic properties is not easy to succeed because of the complexity of direct synthesis. Compared to the direct synthesis, the postmodification or post-functionalization of monoliths certainly represents a complementary and flexible technique for the preparation of monolithic stationary allows complete independent control of the porous properties.

For the separation of complicated biological samples, such as complex mixtures of peptides for proteome analysis and/or enantiomers, monolithic columns with longer lengths, smaller inner diameters, and specialized selectivity to peptides or enantiomers will play a crucial role in hyphenated systems. A new field of study in the field of chromatographic separation science has been made possible by the invention of monolithic stationary phases, which is now playing a considerably more significant role in a wide range of application areas. In comparison to other carriers like inorganic porous solids (mesoporous silica) or organic polymers, MOFs or coordination polymers have many advantages for the adsorption and release of biomolecules because of their tunable composition, structure, pore size, and volume, easy functionalization, flexible network, and/or accessible metal sites. By selecting the right metal, linker, and structure, one can alter the biodegradable properties of these materials, causing a degradation in body fluid that can last anywhere from a few minutes to many weeks. Even if actual porosity "BioMOFs" are still hard to come by, endogenous linker-based MOFs are one of them and are of particular interest. Making a bioactive MOF with the drug as the linker and releasing the drug through the degradation of the MOF is an alternative method of releasing large amounts of drugs. You can also use a bioactive metal (Ag, Zn, Ca, Mn, Gd, Fe, etc.) as the inorganic cation to add extra properties like antibacterial activity or imaging properties.

## **Funding**

The authors extend their appreciation to the Deanship of Scientific Research and the Research Center, College of Pharmacy, King Saud University for financial support.

## **Declaration of competing interest**

The authors declare no conflict of interest.

## **Author details**

Mohamed Hefnawy<sup>1</sup> \*, Ali El Gamal<sup>2</sup> and Manal El-Gendy<sup>1</sup>

1 Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia

2 Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia

\*Address all correspondence to: mhefnawy@ksu.edu.sa

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## **Chapter 4**
