Author's edition

Author's edition

Industrial Applications of Green Solvents

Volume II

Edited by

Inamuddin1,2,3, Rizwana Mobin4 and Abdullah M. Asiri1,2

1
Centre of Excellence for Advanced Materials Research, King Abdulaziz University,
Jeddah 21589, Saudi Arabia
2
Chemistry Department, Faculty of Science, King Abdulaziz University,
Jeddah 21589, Saudi Arabia
3
Department of Applied Chemistry, Faculty of Engineering and Technology,
Aligarh Muslim University, Aligarh-202 002, India
4
Department of Industrial Chemistry, Govt. College for Women, Cluster University, Srinagar,
Jammu and Kashmir-190006, India
 Author's edition
Copyright © 2019 by the authors

Published by Materials Research Forum LLC

Millersville, PA 17551, USA

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Materials Research Foundations
Volume 54 (2019)
ISSN 2471-8890 (Print)
ISSN 2471-8904 (Online)

Print ISBN 978-1-64490-030-7

eBook ISBN 978-1-64490-031-4

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 Author's edition
Table of Contents
Preface

Supercritical CO2 Application in Essential Oil Extraction

Mozaniel Santana de Oliveira, Sebastião Gomes Silva, Jorddy Neves da Cruz,
Eduardo Ortiz, Wanessa Almeida da Costa, Fernanda Wariss Figueiredo Bezerra,
Vânia Maria Borges Cunha, Renato Macedo Cordeiro, et al. .......................................... 1

Applications of Ionic Liquids in Sensors and Biosensors

Amina Saleem, Nawshad Muhammad, Zahoor Ullah, Amir Sada Khan, Abdur Rahim 29

Green Solvents for CO2 Capture

M. Yildiz ......................................................................................................................... 51

Ionic Liquids as Green Solvents for Lignocellulosic Biomass Utilization

H. Mahmood, M. Sulaiman, M. Moniruzzaman ............................................................. 60

Applications of Glycerol as Green Solvent

Harshal D. Kawale, Sneha Acharya, Nanda Kishore ...................................................... 87

Application of Ionic Liquids as a Green Material in

Electrochemical Devices
Alok Kumar Tripathi and Rajendra Kumar Singh ........................................................ 106

Green Chromatographic Purification of Pharmaceuticals

R.N. El-Shaheny, M.H. El-Maghrabey, M.I. Eid, N.M. El-Enany ............................... 148

Water as the Green Solvent in Organic Synthesis

Kartick Chandra Majhi, Paramita Karfa, Sunil Kumar, Rashmi Madhuri ................... 182

Glycerol as a Green Solvent in Organic Reactions

Navneet Kumar, Vimal Chandra Srivastava ................................................................. 202

Ionic Liquids as Green Bio-Lubricant Additives

Puneet Verma, Gaurav Dwivedi, Anoop Kumar Shukla, Anuj Kumar,
Arun Kumar Behura ...................................................................................................... 224

Ionic Liquids as Green Electrolytes for Aluminum and

Aluminum-Alloy Production
Guocai Tian ................................................................................................................... 249

Keyword Index .............................................................................................................. 294

About the Editors........................................................................................................... 295
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Chapter 7

Green Chromatographic Purification of

Pharmaceuticals

R.N. El-Shaheny*, M.H. El-Maghrabey, M.I. Eid, N.M. El-Enany

Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Mansoura
University, Mansoura, 35516, Egypt
* rania_n2010@[Link]
Abstract
Purification of active pharmaceutical ingredients is a crucial step in pharmaceutical
manufacturing. The elimination of by-products, impurities, catalysts, coloring agents, and
residual solvents is critical for the production of high-quality pharmaceutical products.
This step is traditionally time-consuming, expensive, and consumes plentiful toxic
organic solvents. With the global concern about green chemistry and sustainability, it
becomes mandatory for the pharmaceutical industry to depend on greener purification
techniques. This chapter highlights the green chromatographic techniques for purification
of pharmaceuticals including counter-current chromatography, high-performance thin
layer chromatography, gas chromatography, and supercritical fluid chromatography. As
well, the most up-to-date applications of these green chromatographic purification
techniques are included.
Keywords
Supercritical Fluid Chromatography, Gas Chromatography, Countercurrent
Chromatography, Thin Layer Chromatography, Green Purification, Pharmaceuticals

Contents

1. Introduction............................................................................................149
2. Counter-current chromatography .......................................................151
2.1 Introduction......................................................................................151
2.2 Counter-current chromatography as a green purification
tool ...................................................................................................153
2.2.1 Saving of solvents ............................................................................153

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2.2.2 The use of greener solvents .............................................................153

2.2.3 The possibility of injection of crude samples ..................................154
2.2.4 Enhancement of the economic and ecological aspects ....................155
3. High-performance thin-layer chromatography (HPTLC) ................155
3.1 Introduction......................................................................................155
3.2 HPTLC as a green separation tool ...................................................156
3.2.1 RP-HPTLC ......................................................................................157
3.2.2 HILIC-HPTLC .................................................................................157
3.2.3 SO-HPTLC ......................................................................................158
4. Gas Chromatography ............................................................................159
4.1 Introduction......................................................................................159
4.2 Greening sample pretreatment step .................................................159
4.2.1 Pretreatment-free methods (direct methods) ...................................159
4.2.2 Solvent-free sample pretreatment ....................................................161
4.2.3 Green solvents for sample pretreatment for GC ..............................163
4.2.4 Assisted solvent extraction techniques ............................................163
4.3 Greening GC via column and carrier gas optimization ...................165
5. Supercritical fluid chromatography ....................................................166
5.1 Introduction......................................................................................166
5.2 Separation and purification of APIs by SFC ...................................167
5.3 Chiral separation of enantiomers by SFC ........................................168
5.4 Removal of residual solvents from pharmaceuticals .......................169
6. Efforts for greening preparative high-performance liquid
chromatography as a purification tool...........................................................170
Conclusions .......................................................................................................172
References .........................................................................................................173

1. Introduction
The production of active pharmaceutical ingredients (APIs) is the main core of the
pharmaceutical industry. Processes for the production of APIs are chiefly organic
chemistry-based (synthetic pathway), botanically-based, or bio-processing based. In the

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last century, the development of new APIs via organic synthesis pathways has been
greatly contributed to the revolution in medical care. Pharmaceutical products containing
synthetic APIs are very popular due to cost- and time-efficiency, ease of quality control,
and rapid effects. However, in case of synthesis of APIs through multi-step synthetic
pathways, the elimination of reaction by-products, metal catalysts, solvents, colors etc is
essential for the production of high-quality pharmaceuticals [1,2].
In addition, numerous natural product-derived compounds have been applied to drug
discovery and development. Plants have been always a rich source of useful drugs such
as arteether, endoperoxide sesquiterpene lactone, galantamine, dronabinol, cannabidiol,
capsaicin, tiotropium, and apomorphine [3]. Bioprocessing is also a principal source for
the production of APIs. In this technique, natural or genetically-recombinant cells,
tissues, organs, or parts are used for the production of medically or industrially important
products. Examples of medically important compounds produced by this technique are
the DNA for gene therapy and transient infection, antibiotics, proteins (interleukins and
interferons), and hormones (insulin) [4]. Yet, it is essential to consider the isolation of the
APIs produced by these techniques in a pure form.
The separation and purification of APIs are very essential for the pharmaceutical
industry. Yet, there is a challenge to obtain the target compound from the complex matrix
(synthesis mixture, plant/herb, or a bio-processing mixture) in a pure state. Among many
purification techniques, chromatography is still the most widely used purification method
used by most of the leading manufacturing companies for obtaining APIs with high
purity. With this aim, several chromatographic techniques including size-exclusion
chromatography, high-performance liquid chromatography (HPLC), counter-current
chromatography (CCC), thin-layer chromatography (TLC), and supercritical fluid
chromatography (SFC) are currently being used [2-4].
Though, with the widespread use of chromatography as a purification tool in the
pharmaceutical industry, two main problems appeared: the high costs of the large
volumes of organic solvents consumed and the generation of a huge quantity of waste.
These problems are mainly aggravated with the increasing attention received by the
concept of green chemistry. There is a growing interest in the application of the
principles of green chemistry to all chemical researches and industries. The main aspects
of green chemistry are decreasing the consumption of reagents, minimizing the
production of by-products harmful to the human or to the environment, decreasing the
energy consumption, and reducing the costs. However, the value obtained by using
chromatographic techniques for purification of pharmaceuticals overcomes by far the
costs of purchasing solvents and disposing wastes [5]. Yet, there is still a vital question
here: “can we obtain the same results with decreasing organic solvents consumption and

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waste generation?”. Many technological advances that have been recently developed
show that the answer to this question is certainly “Yes”.
Three “Rs” are usually mentioned in connection with green analytical chemistry: reduce,
replace, and recycle. Most efforts for greening chromatographic purification techniques
depend mainly on the reduction of organic solvents consumption and the production of
wastes, replacing the harmful solvents with greener ones, and recycling the wastes [5].
These standpoints are the basics for developing greener chromatographic techniques such
as CCC, gas chromatography (GC), SFC, and high-performance TLC (HPTLC). Also,
some analytical chemists have made efforts for greening preparative HPLC techniques.
Hence, in this chapter, we discuss the modern techniques for greening chromatographic
purification of pharmaceuticals with a special emphasis on the most up-to-date reports
focusing on this subject.

2. Counter-current chromatography

2.1 Introduction
Counter-current chromatography (CCC) is a liquid-liquid chromatography system in
which the solutes are partitioned between two immiscible liquids, one is stagnant (i.e.
stationary phase), while the second liquid passes through (i.e. mobile phase). A schematic
illustration of the basic CCC instrument is presented in Fig. 1. Solutes are separated by
distribution between the two liquid phases. The separation mechanism can be presented
by the following equation:

VR = VM + KD⋅ VS Eq. 1

Where: VR is the retention volume, VM and VS are the mobile and stationary phases
volumes inside the CCC instrument of a total volume VC = VM + VS. KD is the solute
distribution ratio between the mobile phase and the stationary phase which can be
obtained according to Eq. 2 [6]

solute concentration in the stationary phase

KD = Eq. 2
solute concentration in the mobile phase

CCC is a simple and highly productive technique for the purification of complicated
mixtures using solvent mixtures only. Yet, industrial applications of such methodology
are limited to some extent since the separation of solutes can be achieved only by
retention of a large quantity of the stationary phase (VS) in the apparatus. Many designs

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of CCC worked initially by gravity including the droplet CCC columns. Presently,
hydrostatic and hydrodynamic instruments are available and efficiently integrated into
industrial purification of pharmaceuticals [6,7]. In addition, the development of high-
performance CCC allowed its application as a mainstream purification in pharmaceutical
chemistry [8].

Fig. 1. Schematic diagram illustrating CCC, where P and I stand for pump and injector,

The high load ability is the major advantage of CCC, where the interaction of the solutes
with the liquid stationary phase that represents the larger volume of the overall CCC
column volume (70-80 %) permits the injection of a much larger sample amount than in
preparative LC. In preparative LC, the area of the stationary phase surface that is
available for the interaction with the solutes is limited, thus, overloading takes place
rapidly. Additionally, the scaling-up is simple and predictable in CCC since the
separation is controlled only by the distribution between the two immiscible liquids and
the same solvents can be utilized for both small and large columns. In contrary, in
conventional LC, the scaling-up is not direct due to the difficulty of obtaining packing
material of similar characteristics to those used in an analytical column to design a
preparative one.

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2.2 Counter-current chromatography as a green purification tool

2.2.1 Saving of solvents

Chromatographic methods are generally characterized by using large quantities of
organic solvents. In this context, CCC consumes large volumes of solvents, however, the
high capacity resulted in a whole solvent saving. For an explanation of this point, an
example is the purification of the insecticide Spinetoram J from its fermentation by-
product Spinetoram L [9]. The volume of solvents/kg purified substance in case of using
preparative LC is 2560 L/kg (in 400 runs), while it is 733 L/kg purified substance in case
of CCC (9 runs only). Thus, it is obvious that CCC saves solvents (three times lesser
volumes) as well as time. The CCC provides a high throughput of 40g/h Spinetoram J
versus 17g/h for preparative LC. Yet, it should be noted that this comparison cannot be
generalized to all processes.
It is also worthy to consider the possibility of recycling the two immiscible liquid phases
in CCC, especially most procedures are isocratic. This is very advantageous for economic
reasons when dealing with industry. Wu et al. [10] described a recycling CCC method for
purification and separation of phytosterol analogues from the crude extract of green alga
Chlorella vulgaris on the preparative scale. In this method, a biphasic solvent system
consisted of n-hexane/dichloromethane/acetonitrile ([Link], v/v/v) was used with the
lower phase acting as the stationary phase. In the recycling CCC, the effluent is injected
by linking the outlet of the detector to the inlet of the pump and thus limiting the loss of
stationary phase and improving the separation. After 5 runs, 300 mg of crude C. vulgaris
extract gave rise to 20.3 mg ergosterol and 11.7 mg 26-nor-25-isopropyl-5,7,22-trien-3β-
ol. This technique has been promising for large scale applications in industry.

2.2.2 The use of greener solvents

Water, the greenest and most available solvent, is very commonly used in CCC for
purification of polar compounds. However, chlorinated solvents are also very common in
CCC. The ternary mixture of chloroform/methanol/water is very popular in CCC for
compounds with median polarity, but this system has very hazardous and toxic impacts
on the environment and the operators. The European Registration, Evaluation,
Authorization and Restriction of Chemical [11] has recommended limiting its use. As
substitutes, esters, ethers, ketones, and nitriles have been recommended due to the
similarity in polarity. But, in contrast to the chlorinated solvents, the densities of all these
solvents are less than water and thus constitute an upper organic layer.
Some options have been also introduced as greener solvents for CCC. Chen and co-
workers have designed an online two dimensional (2D) system consisted of a CCC and

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preparative HPLC for purification and separation of polar polyphenols from tea extract
[12]. The CCC employed a harmless and eco-friendly biphasic solvent system comprising
isopropanol and 16% sodium chloride aqueous solution (1:1.2, v/v). After fractionation
by the CCC, the effluent was purified by octadecyl silica-packed column as the second
step. This system allows identification and preparative separation of ten polar solutes
from aqueous Chinese oolong tea extract on a large scale via a single step which is
promising for drug discovery.
Ionic liquids (ILs) are also highly recommended greener solvents for CCC applications.
ILs are salts in which the ions are poorly coordinated and thus they exist as liquids at a
temperature less than 100°C or even at room temperature. They are non-volatile, so, the
risk of evaporation to the atmosphere and subsequent toxicity are minimized [13].
Recent researches focused on studying the hydrodynamic behavior and retention of
biphasic ILs in CCC employing high-speed imaging, providing a good understanding of
CCC chromatographic extraction using ILs [14]. An IL-modified high-speed CCC
method was recently established by Wu et al. [15] for the separation of four alkaloids
(pronuciferine, N-nornuciferine, nuciferine, and roemerine) from crude extract of Lotus
leaves with high purity (˃90.5%). The presence of IL,1-butyl-3-methylimidazolium
tetrafluoroborate improved separation efficiency and speed.

2.2.3 The possibility of injection of crude samples

Since the mobile and the stationary phases are two liquids in CCC and no risk of clogging
exists, the direct injection of concentrated and crude samples is feasible. The sample may
be dissolved in one of the two phases or in both of them. This characteristic provides an
excellent advantage for injection of crude samples such as plant extracts with minimum
pretreatment steps, thus, augmenting the CCC greenness. In contrast, this is not probable
in case of preparative LC where the concentrated samples may cause clogging of the
column due to particulate matters.
A new approach involved a preparative fast speed CCC procedure together with direct
injection of a powder sample was applied for separation and purification of four alkaloids
(jatrorrhizine, coptisine, palmatine, and berberine) from Coptis chinensis Franch without
any sample pretreatment steps and with higher recovery (˃92%) and purity than ordinary
extraction methods [16]. Another example is the direct injection of a crude extract of
Herba Salviae Plbeiae dissolved in the mobile phase coupled with high-speed CCC for
the separation and purification of four flavonoids (hispidulin, nepetin, homoplantaginin,
and nepetin-7-glucoside) on a preparative scale using dual elution mode [17]. This
technique obviously reserved solvents and time and thus, conserved the environment.

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2.2.4 Enhancement of the economic and ecological aspects

Improvement of the greenness of CCC can be achieved by some strategies such as
multiple injections [18] and dual mode or multiple dual-mode [19]. The multiple
injections technique depends on the injection while the previous run is still on. This
technique increased the throughput of CCC. The dual mode technique depends on
swapping the phase role during the separation process since any of the two liquid phases
can be employed as the stationary phase. The multiple dual mode depends on repeating
this swapping several times during the separation. In this strategy, the solutes which
partitioned into the upper phase are eluted at an end of the CCC tubing, while the solutes
partitioned into the lower phase are eluted at the second end. The switching of phases
causes moving of the solutes up and down and virtually increases the tubing length and
permitting the complete separation of solutes [19].
Bradow et al. constructed a fully-automated CCC system for fast screening of the
quaternary solvent mixtures (heptane/ethyl acetate/methanol/water) and translation of the
outcomes to separations on the preparative scale. This approach allowed decreasing the
screening time to about 17 min for each solvent system and consequently speeding the
purification procedure. The purification of a synthetic tetrazole epoxide produced by a
medicinal chemistry support workflow has been performed via mass-directed preparative
CCC [20].
In addition, many processes have been implemented for the improvement of the
separation power and detection potential of CCC such as high-speed CCC, 2D-CCC,
multidimensional CCC, and online coupling with liquid chromatography [21]. These
procedures help to enhance the economic and ecologic features of CCC, thus, making it
greener for pharmaceutical purification.
Thus, by virtue of the great advances in the CCC models which reached maturity now as
well as the possibility of greening the technique to a high extent, a marvelous growth in
its applications in pharmaceutical industries is expected in the future.

3. High-performance thin-layer chromatography (HPTLC)

3.1 Introduction
TLC is a liquid chromatography method that has the same separation principles of HPLC
but it differs in the configuration of the stationary phase. The stationary phase in TLC is a
solid porous substance supported on a solid support (frequently glass, plastic, or metal),
thus, exhibiting a relatively large surface area. The mobile phase is a liquid consisting of
one or more solvents. The chromatographic separation in TLC is controlled by the

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interaction forces including the interaction of solutes with both of the stationary and the
mobile phases as well as the interaction of the mobile phase and the stationary phase
themselves. These interaction forces embrace hydrogen-bonding, charge transfer,
electrostatic attraction, and van der Waals forces [22-24].
HPTLC is an innovative form of TLC which involves utilizing a high-performance
stationary phase, an implemented instrument, and validated methods. HPTLC is
characterized by comparable accuracy and precision to HPLC and GC techniques while
having many advantages over them such as the need for a small sample volume, simple
and cheap instruments, possibility of separation of many samples on the same plate
offering higher throughput and lower costs, facile optimization, consumption of smaller
volume of mobile phase, time-saving, and cost-efficiency [24].
Further impressive progress in liquid chromatography has led to the development of
monolithic materials as stationary phases. With this development, the ultrahigh-
performance thin layer chromatography (UPTLC) has been evolved by using monolithic
or nanostructured sorbents. With this technique, the mobile phase consumption decreased
by several folds, and the sensitivity is significantly enhanced. Yet, the resolution is
adversely affected by virtue of the shorter development time and the availability of
smaller surface area [25]. Thus, the UPTLC is considered greener than the HPTLC,
however, the latter is a more applied chromatographic technique.

3.2 HPTLC as a green separation tool

The main applications of TLC within the framework of the pharmaceutical industry are
mainly concerned with the drug discovery and development process. It is also used to
obtain information about purity prior the transfer from analytical to preparative LC to
decrease the number of LC trials needed to achieve this aim, thus, decreasing the
consumption of solvents, time, and efforts. Though the modern HPTLC techniques are
inherently greener than HPLC, their greenness can be significantly enhanced in the light
of the three Rs concept. Most of the HPTLC procedures adopt normal phase (NP) mode
using a stationary phase more polar than the mobile phase. The common solvents used
for mobile phase preparation, such as hexane, dioxane, formic acid, chloroform, diethyl
ether, and benzene, are very toxic. In consequence, some separation modes of HPTLC
adopted the replacement of these solvents with safer and greener ones such as water,
alcohols, and water/salt mixtures. Three techniques are currently widespread: reversed
phase HPTLC (RP-HPTLC), hydrophilic interaction liquid chromatography-HPTLC
(HILIC-HPTLC), and salting-out-HPTLC (SO-HPTLC) [26]. The bases and some recent
applications of these techniques are being discussed below.

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3.2.1 RP-HPTLC
In this separation mode, the polarity of the stationary phase is less than that of the mobile
phase. This can be achieved by partial alteration of silica gel surface through binding of
the siloxane groups with alkyl ligand such as ethyl, octyl, dodecyl, octadecyl, or phenyl.
The partial modification allowed good wetting of the stationary phase with the polar
mobile phase and offered the RP nature to the stationary phase. The mobile phases
frequently used with this mode are mixtures of water and organic modifiers of less
toxicity than ordinary HPTLC organic solvents, such as methanol, acetonitrile, and
acetone. Moreover, these solvents could be replaced with ethanol which is a greener
solvent [26].
In this context, Gabriëls and Plaizier-Vercammen [27] designed RP-HPTLC and NP-
HPTLC methods for separation of different groups of analytes that have different
polarities including artemisinin, artesunate, artelinic acid, arteether, α and β isomers of
artemether, dihydroartemisinin, and desoxyartemisinin, and some of their decomposition
products. RP-C18 F254S and silica gel 60 F254 TLC plates were used and the mobile
phases were chloroform-methanol (70:0, 60:10, and 50:20 v/v) for the NP mode and
methanol-water (50:20 v/v) or acetonitrile-water (50:20 v/v) for the RP mode. This
method was superior to the HPLC method reported by Molnár [28] which was not
adequate to resolve all compounds. In addition, the HPTLC method offered the superb
ability for stability examination of these compounds, their degradation products, and
impurities.

3.2.2 HILIC-HPTLC
HILIC is a mode of liquid chromatography well-suited for separation of highly polar
compounds which suffer from poor retention by RP-HPLC. The stationary phases used in
HILIC include silica, cyanopropyl, diol, triazole, as well as zwitterion stationary phases
and the mobile phase is a mixture of organic solvent (acetonitrile or ethanol) and water.
Multiple separation mechanisms including ion exchange and partitioning are involved in
HILIC [29].
Zheng et al. [30] designed hydrophobic-hydrophilic monolithic dual-phase plates via
two-step polymerization procedure for 2-dimensional (2D) TLC of several dyes with
diverse polarities. The dual-phase layer offered two areas with very different polarities,
thus, including HILIC and RP separation approaches. The mobile phases were ethyl
acetate/ethanol/water [Link] v/v/v (RP dimension) and10 mM NaCl solution in methanol
(HILIC dimension). These mobile phases are greener than those commonly used for
conventional TLC methods.

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Another example of the application of HILIC-HPTLC is the separation of benzophenone-

4 from components of shampoo using silica gel 60 sorbent and a mobile phase containing
ethyl acetate-ethanol-water-phosphate buffer (pH 6) ([Link] v/v/v/v) with densitometric
detection [31]. The greenness of the mobile phase is also evident in this method.

3.2.3 SO-HPTLC
In SO-HPTLC, concentrated solution of inorganic salts (mostly ammonium sulfate) as
mobile phases with a polar stationary phase (silica, polyamide-, or cellulose-modified
silica) are used. The high concentration of the salt causes a decrease of the solubility of
non-ionized compounds, so, they interact strongly with the stationary phase and strongly
retained, this mechanism is called hydrophobic interaction. These mobile phases are
obviously very safe and hazardless in respect to those used in conventional HPTLC.
Mohamed and colleagues [32] used a SO-TLC technique to elucidate the quantitative
structure-retention relationship of certain oral hypoglycemic compounds including
glibenclamide, glimepiride, gliclazide, glipizide, repaglinide, pioglitazone HCl, and
metformin. Silica gel 60 plates and a mobile phase composed of ammonium sulfate-
acetonitrile (7:3 v/v) mixture were employed as TLC system. The results of this study
showed that the most significant factors governing the retention include log P(o/w), Van
der Waals force, molar refractivity, hydrophobic surface area and volume, and the
solvation energy. Further, the same research group developed a SO-TLC procedure for
stability testing and concurrent estimation of glimepiride and metformin [33].
As well, Ciura et al. [34] developed a SO-TLC method to investigate the lipophilicity and
hydrophobicity parameters of nine macrolide antibiotics. The obtained chromatographic
data are significantly correlated to the calculated lipophilicity. Quantitative structure-
retention and structure-activity relationships equations were derived. This evidenced that
the results attained by SO-TLC were useful in predicting the antimicrobial activity of
these compounds.
From the aforementioned discussion, we can conclude that TLC will continue to play a
chief role in the pharmaceutical industry with a wide field of applications especially in
drug discovery, purification, and isolation processes in addition to analytical applications.
TLC also offers important information about purity during the scaling-up step, thus,
decreasing the number of the needed chromatographic runs and conserving organic
solvents. The use of more environmentally friend mobile phases as in cases of RP-
HPTLC, HILIC-HPTLC, and SO-HPTLC obviously makes the process greener and safer.

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4. Gas Chromatography

4.1 Introduction
GC is also a common chromatographic technique that is frequently used for the analysis
and purification of pharmaceuticals. This technique utilizes a liquid stationary phase and
a gas mobile phase. The most frequently applied gas as a mobile phase for GC is helium
(90% of the applications) which is an inert gas as well. Some applications involved
nitrogen as a non-reactive gas while the stationary phase is a film of liquid supported on a
glass or metal tubing. The analytes, which should be vaporized without decomposition,
partitioned between the liquid stationary phase and the gaseous mobile phase [35].
Despite GC being inherently greener than HPLC, there are some major challenges for
greening GC procedures for purification of pharmaceuticals. The need for liquid-liquid or
solid-liquid extraction for sample preparation and derivatization process prior GC are the
major problems since these steps consume huge quantities of costly and harmful organic
solvents, in addition to generation of a large volume of waste. In consequence, the most
important aspect to be considered for greening GC is the sample preparation. In addition,
more eco-friendly GC procedures can be achieved by considering the column and the
carrier gas to decrease the analysis time, increase the sample throughput and decrease the
costs of the analysis [36].
It is worth noting that many of the applications reported in the literature for greening GC
procedures were concerned with fields other than the pharmaceutical industry, though,
the adopted principles can be efficiently extended to pharmaceutical applications. The
most prominent role for GC in the pharmaceutical industry involves the detection and
removal of residual solvents as well as impurities from APIs and pharmaceuticals. In
view of the previous aspects, the most modern approaches for greening GC techniques
will be discussed below.

4.2 Greening sample pretreatment step

4.2.1 Pretreatment-free methods (direct methods)

The direct and pretreatment-free GC procedure would be very advantageous for greening
this technique by minimizing the consumption of organic solvents and waste production
and consequently decreasing the costs and harmful impacts of these solvents. Two
sampling approaches are currently applied for this aim: headspace and direct injection. In
headspace GC, the volatile sample components are diffused into space above the sample
when the sample is heated to a moderate temperature in a sealed vial (Fig. 2). Water or a
high-boiling point solvent may be added to the sample to facilitate the dissolution and the

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release of the volatile compounds to the headspace. This technique is very appropriate for
the investigation of residual solvents in pharmaceuticals, flavors in food and drinks, and
monomers in polymers [37]. On the other hand, direct injection GC entails introducing
the whole sample into a vaporization chamber where it is vaporized and transferred by
the carrier gas into the column. Programmed temperature vaporizer (PTV) injector is also
used for introduction of the sample at a low temperature and after injection, the
temperature of the PTV is elevated to vaporize the sample into the column. This mode of
injection has been suitable for the determination of fatty acids, pesticides, and oils in food
[38].

Fig. 2. Illustration of headspace injector for GC

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Nacham et al. [39] have recently studied the use of ILs as diluents for headspace GC to
estimate the residual solvents in pharmaceuticals containing quinidine and indomethacin.
1-Butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide and
trihexyltetradecylphosphonium bis[(trifluoromethyl)sulfonyl]imide were both
investigated and the former was found superior since the sensitivity for the residual
solvents was markedly better due to its negligible vapor pressure and excellent solvation
properties. ILs are considered alternate greener solvents to be used instead of common
organic solvents, where low vapor pressure and high thermal stability are advantageous.
In addition, the use of ILs is economic for industrial purposes [40]. Hence, the use of ILs
for headspace GC significantly improves the greenness of the technique and greatly
enhances the sensitivity which is a major concern in headspace GC. A 25-fold
enhancement of sensitivity was achieved via ILs-headspace compared to headspace
employing convenient diluents [41].
Recently, a new sample introduction approach has been designed by Poronsky and
Cutrone using chromatoprobe thermal extraction device as an alternative to direct sample
injection method. The chromatoprobe thermal extraction offered many advantages over
the direct sample injection technique for estimation of residual solvents in
pharmaceuticals. It consumes markedly smaller amount of the sample (<1 mg), offers
greater sensitivity, overcomes the interference from the diluent, permits the analysis of
compounds with poor solubility in dimethyl sulfoxide, and it is specific for volatile
components of the sample [42]

4.2.2 Solvent-free sample pretreatment

Some samples are not suited for direct GC analysis such as biological matrices and some
environmental samples. Thus, sample pretreatment is mandatory in such cases. Yet, the
ordinary extraction techniques are disadvantageous as discussed earlier. Hence, there is a
strong need for extraction methods that decrease or completely exclude the use of
hazardous organic solvents while keeping the extraction steps minimal, so-called;
solvent-free techniques. This concept minimizes the environmental pollution by the
wastes, decreases the costs of the procedure, and avoids the exposure of workers to toxic
and harmful solvents [36]. The solvent-free extraction methods for GC analysis are
categorized into four types; solid-phase extraction (SPE), thermal desorption (TD),
vapor-phase extraction (VPE), and membrane extraction (ME) [41].
The idea of SPE includes the distribution of the analyte between its matrix and a sorbent,
then extraction of the analyte into a non-miscible phase. Several SPE techniques include;
open-tubular trapping, solid phase microextraction, stir bar sorptive extraction, gum-
phase extraction, closed-loop stripping analysis, and sorption tubes [41]. The

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combination of SPE and TD of the analytes offered a great advantage of elimination of

organic solvents. In TD, the analytes are extracted from the matrix or from the retaining
sorbent by means of heat and inert gas. The gas carries the sample to the analyzer. TD
offered many merits over the conventional liquid-liquid extraction such as greater
sensitivity, the need of a small amount of sample, cost efficiency, elimination of solvents,
full automation, and avoiding solvent interference with the analysis and solvent selection
problems [43]. Though, some drawbacks are associated with TD since the high
temperature may cause degradation, molecular rearrangement of the analyte, or carry
over [44].
Thermal SPE coupled with TD-GC-mass spectrometry (MS) has been applied by
Duemichen and Braun for detection of polymers during thermal and thermo-oxidative
degradation. The degradation products were adsorbed out of the thermogravimetry
(TGA) on small stir bars (called twisters) having a definite quantity of surface adsorption
sites. Then, analysis of the twisters was performed via TD-GC-MS. This method offered
the merits of the TGA for thermal extraction, facile sample preparation, mass detection
ability, and the use of inert and oxidizing atmosphere [45].
In VPE, the analyte is moved from a vapour to a solid phase by passing the vaporized
sample through a stationary phase followed by desorption by the carrier gas. In this
connection, a VPE-GC-MS method was developed to quantify salicylic acid and
jasmonic acid in plants. This method relied on a single extraction step, phase partitioning,
methylation with HCl/methanol, and collection of volatilized methylated analytes on
Super Q filters, thus, eliminating other purification steps. This method offered high
sample throughput and prevented its loss of analytes by omitting multiple sample
cleaning steps [46].
ME is based on the partitioning of the analyte between the sample matrix and the
membrane material. Many membrane materials and configurations (film, hollow fiber)
are available to extract many compounds from gas and liquid matrices. The membrane is
located in contact with the sample (gas or liquid) from one side, then a gas stream is
passed through it and carries the analyte to the analyzer. Thus, ME technique minimizes
the consumption of organic solvent to a high extent. Currently, several advances were
implemented in the field of ME such as microporous membrane liquid-liquid extraction,
extracting syringe, two-phase hollow-fiber-protected liquid-phase microextraction and its
modifications, and membrane extraction with sorbent interface and its variants [47].
In this connection, Al-Zahrani et al. [48] developed a membrane-assisted concurrent
extraction and derivatization method for elemental sulfur in Arabian crude oil by GC-
MS. In this method, a porous hollow fiber membrane filled with the derivatizing

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agent(triphenylphosphine) was utilized as a solvent bar tumbled freely in the crude oil
sample to extract and derivatize the elemental sulfur. The single-step for simultaneous
derivatization, extraction, and preconcentration offered time and cost efficiencies and
omitted the consumption of solvents.

4.2.3 Green solvents for sample pretreatment for GC

Another trend for greening the GC procedures is carrying out the sample preparation
steps using greener solvents. The techniques employed for this purpose include;
supercritical fluid extraction (SFE), subcritical water extraction (SWE), cloud-point
extraction (CPE), and ILs [41].
SFE utilizes non-toxic CO2 which offered many advantages such as safety, non-toxicity,
cost-effectiveness, ability to dissolve many compounds, availability at high purity, and
low critical parameters. SFE enables the extraction of organic pollutants from
environmental samples and solvents residues from pharmaceuticals. On the other hand,
the SWE is dependent on using pressurized hot water as extraction system. The use of
water which is the ideal green solvent is very advantageous in terms of availability, cost-
effectiveness, and eco-friendship [41].
CPE depends on the separation of aqueous micellar solution into two isotropic phases by
controlling the temperature, pressure, or by the addition of a certain substance. The two
phases are a surfactant phase with a smaller volume which trapped the target compound
and a bulk aqueous [Link] is a green technique since the surfactants are cheap, non-
flammable, non-toxic, non-volatile, and biodegradable. This technique is very suited for
extraction of metallic species [49]. Meanwhile, ILs offered high solvation power for
organic and inorganic compounds in addition to their greenness as discussed earlier.
Thus, ILs are applied as green solvents for sample extraction prior to GC [50].
Table 1 shows some applications of GC coupled with the four discussed extraction
techniques relied on green solvents [51-54]. Though most applications were performed
on the lab-scale, the scaling-up has been efficiently studied by Islam et al. [53] proving
SWE as a promising extraction technique for disposal of soil contaminated with
pesticides.

4.2.4 Assisted solvent extraction techniques

Three main techniques widely used to assist solvent extraction while decreasing the
solvent consumption and saving time and efforts are microwave-assisted extraction
(MAE), ultrasound-assisted extraction (UAE), and pressurized-liquid extraction (PLE). In
MAE, the microwave energy irradiates the sample exists in hexane as a microwave

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transparent solvent at controlled temperature and pressure [55]. Whereas in UAE, the
extraction is aided by ultrasound energy at controlled temperature and ultrasound power
[56]. Meanwhile, PLE depends on extraction using solvents at high temperature and
pressure for enhancing the solubilization and mass transfer powers. If the solvent used is
water, the technique is referred to as SWE [57].

Table 1. Some applications of GC coupled with extraction techniques employing green

solvents
Technique Analyte matrix Extraction media Analytical Ref
conditions
SFE-GC- Moderately volatile Pharmaceutical Supercritical CO2 CPSil 24 51
MS impurities tablet preparations at 1.5 mL/min, the CB
of the pressure values in stationary
benzodiazepine the extraction phase,
series(diazepam, system varied programmed
phenazepam, from 80 to 250 column
nitrazepam, and atm at a constant temperature,
clonazepam) temperature of helium as
40°C carrier gas
SWE-GC- 21 Organochlorine Black tea Target DB-5 52
MS/MS and pyrethroid compounds were capillary
pesticides extracted with column,
subcritical water
at 150°C for 15
min, transferred
into acetone-n-
hexane (1:1, v/v),
and cleaned-up by
SPE
SWE-GC- Pesticide (diazinon, Oils Subcritical water Programme 53
MS/MS parathion, (pressure: 1, 2, 3 d column
phenthoate, EPN) MPa, temperature: temperature,
75, 100, 125, 150 helium as
°C) carrier gas
CPE-GC- Haloanisoles (2,4,6 Alcoholic 0.2 g NaCl - 0.5 HP-5MS 54
MS with trichloroanisole, beverages mL phosphate stationary
direct 2,4,6- buffer solution phase,
microvial tribromoanisole, (0.2 M, pH 7.2), programmed
insert 2,3,4,6- 100 µL of 30% column
thermal tetrachloroanisole (w/v) Triton X- temperature
desorption and 114, 75 °C in a helium as
pentachloroanisole) microwave oven carrier gas
for 40 s

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These three approaches are green by virtue of the need for small solvent volumes,
decreased energy consumption, decreased extraction time, and high extraction efficiency.
Yet, the MAE and UAE are suitable for thermolabile compounds, while in case of the
PLE, caution is required to prevent degradation of such compounds by the elevated
temperature. Another limitation of PLE is the expensive instrumentation needed, while
those for MAE and UAE are cheaper [55-57].

4.3 Greening GC via column and carrier gas optimization

The columns used for GC applications may be packed or capillary columns. Table 2
shows the main characteristics of both columns. Greening the GC procedures can be
accomplished via shortening the analysis time and consequently enhancing the sample
throughput and decreasing the costs. Reduction in the analysis time can be achieved by
decreasing the length of the packed column while using packing with smaller particle
size, which increases the column efficiency. On the other hand, the capillary columns are
more popular than the packed columns for most GC applications due to a higher speed,
better resolution in addition to their inherent greenness [58].

Table 2. The main differences between the packed and capillary columns for GC
Parameter Packed column Capillary column
Length (m) 1-5 10-100
Internal diameter (mm) 5 0.25
Material Stainless steel or glass packed Fused silica with its inner
with the stationary phase or side coated with the
inert pack coated with the stationary phase
stationary phase

Decreasing the time needed for analysis in GC can be also achieved by increasing the
velocity of the carrier gas. The most employed carrier gases for GC methods are helium,
nitrogen, and hydrogen. Hydrogen is the carrier gas with the minimum viscosity, thereby
having the highest velocity at a given pressure. Thus, the use of hydrogen as a carrier gas
decreases the analysis time. Yet, helium is more commonly used than hydrogen. But, its
availability in high purity has become a problem. On the other hand, hydrogen produced
from water has high purity and easy availability [59].
In this context, some recent researches were reported encouraging more applications of
GC using hydrogen as a carrier gas. Lin and researchers [60] group utilized hydrogen as a
carrier gas for the recognition of adulterant in traditional Chinese medicine and food
supplements. Utilizing hydrogen or helium as a carrier gas gave rise to the same limit of
detection for 170 drugs with satisfactory analysis of real samples in both cases.

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Additionally, the analysis time was reduced. Polychlorodibenzo-p-dioxins and

polychlorodibenzo furans have been determined in stationary source emissions by GC
with hydrogen as a carrier gas. The runtimes were reduced by about 35% while having
excellent robustness and accuracy. This significantly decreased the waste of time, money,
and efforts [61].
In conclusion, although LC is more predominant in pharmaceutical analysis and
purifications, GC still preferred over it in the industrial analytical laboratories. The
simplicity of operation, robustness, ease of automation, the cheapness of the gas, high
sample throughputs and speed of analysis are all characteristics of GC that are also chief
benchmarks to the industrial analytical laboratories. Furthermore, the inherent greenness
of GC relative to LC as well as the availability of many greening approaches gives it a
superb advantage. The most prominent application of GC in the pharmaceutical industry
is residual solvents elimination during the manufacturing of drug substances as well as
detection of impurities. As discussed in this section, many fertile ideas are available for
greening GC methods suited for chromatographic purification.

5. Supercritical fluid chromatography

5.1 Introduction
Supercritical fluids are gases or liquids at temperature and pressure above their critical
points (Fig. 3). These are characterized by having densities closer to liquids and viscosity
similar to gases, and not possessing a surface tension since there is no distinction between
gas and liquid phases. Thus, supercritical fluids have greater solvation power than gases
and better diffusivity and flow than liquids [62].

Fig. 3. A single component phase diagram

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SFC was suggested for the first time by Lovelock in 1958 and the first SFC apparatus
was designed by Hewlett-Packard during 1981-1982. In SFC, the mobile phase is a
supercritical fluid compressed and pushed through the column. Prior delivery to the
column, its temperature is elevated to exceed the critical temperature and the pressure
within the column is fixed. In SFC, both open tubular and packed columns are used. The
packed columns are similar to those used for HPLC and are more popular for modern
applications of SFC. Silica or alumina columns are used for compounds of low polarity
while alkyl-bonded phases are used for polar compounds. The most commonly used gas
for preparation of the mobile phase is CO2 which has critical temperature and pressure of
304.1 °K and 73.8 Pa, respectively. A polar modifier is frequently mixed with the mobile
phase in order to increase the solubility of polar compounds, and decrease the retention
volume. The organic modifier must be miscible with the supercritical fluid. Methanol,
ethanol, n-propanol, isopropanol, and water are the commonly applied polar modifiers
[41, 62]. The supercritical CO2 is a potent green solvent by virtue of its non-flammability,
non-toxicity, and feasibility of its removal by depressurization. In addition, its recycling
is also possible in an environmentally safe way [62]. Thus, SFC has many unique
advantages that make it green and eco-friendly chromatographic technique. It excludes
the use of organic solvents, instead, safe CO2 is used as a green solvent. As well, SFC
offers fast separation, low costs, and high resolution at low temperature. Yet, the need for
sustaining a constant pressure during the analysis and the time needed for cleaning are
two limitations for SFC [41].
Today, SFC is a powerful separation and purification tool in various industrial areas such
as the food industry, material and polymer science, thermal and nuclear power
generation, and the pharmaceutical industry. SFC has excellent capabilities on the
analytical, semi-preparative, and preparative scales. Consequently, it finds many
applications in all stages of the pharmaceutical industry. The main applications of SFC in
pharmaceutical purification will be discussed below.

5.2 Separation and purification of APIs by SFC

Nowadays, SFC is abundantly used for separation and purification of API from
production matrices, plant or herb, or from by-products. Cyclosporine A produced by
biotechnology from Topylocladium inflatum, has been purified by preparative SFC using
moderate temperature, silica column, and CO2 as a supercritical fluid mobile phase and
adopting a recycling procedure. The industrial setup produced Cyclosporine A with a
purity of 98-99% which satisfied the requirements of US Pharmacopoeia [63] after
[Link] has been applied also for removal of surface-bound impurities

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(formic acid and formaldehyde) from pharmaceutical excipients (starch, microcrystalline

cellulose, hydroxy-propylcellulose, polyethylene oxide, and poly-vinylpyrrolidone [64].

5.3 Chiral separation of enantiomers by SFC

SFC becomes the main tool for chiral separation of pharmaceutical enantiomers on
analytical and preparative scales. There are two main challenges associated with
preparative SFC: the first is the need for huge amounts of CO2, and the second is the
collection of the target compound. Regarding the first challenge, recycling of CO2 is the
solution which decreases the consumption of fresh CO2 to a high extent. This could be
achieved by cooling CO2 at 45 Pa with special consideration for the removal of the
modifier from CO2 by using a flow of liquid CO2. The challenge associated with the
solute collection is the increase of the mobile phase volume at the step of
depressurization to separate CO2, modifier, and the compound of interest which reaches
500-folds expansion. This causes re-mixing of the phases as well as the formation of
aerosol at the workplace and decreases the purity and the yield of the target compound.
Some methodologies have been adopted to overwhelm this problem, for instance,
pressurized fraction collection by cyclone and phase separator to govern the conversion
of CO2 from supercritical state to gaseous state via depressurization without production of
a spray. The latter method allowed removal and collection of modifier at very low
pressure and it was the base for further improvements achieved by Waters Company to
design preparative 100 SFC MS device which has been applied efficiently to purify
compound libraries with a throughput similar to preparative LC methods [65].
The chiral separation of enantiomers can be achieved by one of two methodologies:
direct and indirect methods. The direct method involves the use of a chiral stationary
phase to produce temporary diastereoisomers between the target compound and the
stationary phase, while in the indirect method the target compounds are derivatized using
a chiral derivatizing agent or a chiral additive added into the mobile phase, therefore,
these compounds could be separated with achiral stationary phase. The second approach
is more economic regarding the costs of the chiral stationary phase which is highly
expensive, but it also required costly chiral derivatizing agents or additives which must
have high enantiomeric purity, in addition to the time needed for removal of the chiral
agent from the target compound. In consequence, the direct approach is the one applied
for preparative chiral SFC allowing the application to a wide variety of compounds and
obtaining enantiomers with high purity on a large scale. The utmost widespread chiral
stationary phases are the polysaccharide-derived selector adsorbed on silica since they
can resolve a variety of racemates and a good loadability can be gained for preparative
applications [65].

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An example of preparative chiral separation employing SFC is the purification of

flavanone (Fig. 4) by employing Alcyon SFC CSP Amylose-C column and CO2/ethanol
(80/20) eluent at 30°C/15 mPa. The yield of SFC was twice that of preparative HPLC
(using n-hexane/ethanol (90/10) eluent and CHIRAL ART Amylose-C columns) along
with the fraction volume (L solvent/g product) lesser than that of HPLC. As well, the
solvent consumption reduced by about 25% and the total cost of SFC was approximately
50% of that in the case of HPLC [66]. In addition, ethanol used in the SFC method is the
benchmark green solvent while hexane used for the HPLC method was highly toxic and
hazardous. Another example of preparative chiral SFC has been introduced by Nie et al.
[67] for the improvement of the chiral separation of (R, S)-goitrin (Fig. 4). SFC was
adopted on a Chiralpak IC column by means of Co2/acetonitrile (80/20) as the eluent at
35°C/100 bar. This method offered a 10-times enhancement in the rapidity relative to the
reported normal phase HPLC method. The yield of the SFC preparation exceeded 90%,
based on the total amount of the two isomers, with chiral purity greater than 99%.

Fig. 4. Chemical structures of (a) (R) and (S) flavanone and(b) (R) and (S) goitrin

5.4 Removal of residual solvents from pharmaceuticals

Organic solvents are constantly consumed at different stages during pharmaceutical
manufacturing of the APIs and preparation of the pharmaceutical products. Organic
solvents are not completely removed from the APIs following the usual manufacturing
processes. The remaining solvents are commonly referred to as “residual solvents”. Since
the exposure to organic solvents involves many health hazards such as neurotoxicity,

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liver, kidney and respiratory disorders, and dermatitis, these must be completely removed
from the pharmaceuticals. Currently, residual solvent control has been adopted in the
United States Pharmacopoeia (USP) [68], the British Pharmacopoeia (BP) [69], the
European Pharmacopeia (Ph. Eur.) [70] and the Chinese Pharmacopoeia [71].
Various methods reported for the removal of residual organic solvents include the use of
supercritical fluid extraction technique rather than SFC. Casali et al. [72] described a
technique for the removal of more than 95 % of residual glutaraldehyde from crosslinked
collagen films via contact with supercritical CO2 for one hour. A procedure was also
described for the simultaneous removal of organic solvents and sterilization directly in
dispensing vials using supercritical CO2 and a sterilant. The model drugs were
acetaminophen and paclitaxel. The method was carried out at 37 °C (±2 °C), 8 mPa and
90 min full cycle time. The recovery of the two drugs was 100 % (±0.5 %) and the
procedure has no effect on their stability. This technique has been promising particularly
for the drugs vulnerable to hydrolysis in the presence of water [73].
It is worthy to note that, the supercritical fluid technologies have found many applications
in different areas of modern pharmaceutical industries including their applicability for the
micronization of many drugs such as atorvastatin calcium, lysozyme, 5-fluorouracil, and
cyclosporine A. Supercritical fluid technologies are used also for the production of solid
dispersions free from organic solvents for solubility enhancement of drugs such as PEG
4000-based solid dispersion of carbamazepine. Another important usage of supercritical
fluids has been the manufacturing of micro and nanoparticles. Microparticles of naproxen
and lovastatin, as well as nanoparticles of gentamycin and naltrexone, have been
produced by this technique [74]. An additional interesting application of supercritical
fluid technologies was the production of pharmaceutical co-crystals which are a novel
crystalline form of APIs for the improvement of solubility or stability. Examples of co-
crystals prepared by supercritical fluid technologies include piracetam–salicylic acid,
naproxen–nicotinamide, and carbamazepine–saccharin co-crystals [75]. Also,
supercritical fluid technologies have been used for drying at different stages of
pharmaceutical productions without affecting the solid particles appearance [74].

6. Efforts for greening preparative high-performance liquid chromatography as a

purification tool
Most efforts for greening HPLC techniques have been concentrated on the analytical
separations, and a comparatively little consideration has been focused on the preparative
applications. This is attributed to the difficulty of making them greener since preparative
HPLC excludes the utilization of some additives as in case of micellar liquid
chromatography or the employment of green solvents with high viscosity like propylene

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carbonate. Yet, some efforts are still continued for greening preparative HPLC methods
for purification and industrial purposes mostly via recycling of the mobile phase or
replacement of toxic solvents with greener ones.
In this context, Dhanarajan et al. [76] adopted a new strategy for the designing and
scaling-up of a green HPLC technique without solvent waste that permitted the
purification of three antimicrobial lipopeptide families (iturin, fengycin, and surfactin)
produced by marine Bacillus megaterium rapidly with high efficiency. The HPLC waste
was treated via fractional distillation to recover acetonitrile. Then acetonitrile was filtered
through a 0.22 μm PTFE membrane prior reusing. The UV absorbance of the recovered
acetonitrile was similar to that of the pure unused solvent. This treatment strategy
completely avoided waste production. The best recoveries gained for the surfactin, iturin,
and fengycin families were 96, 95, and 93%, respectively, with purities of 97, 96, and
95%, respectively. The outputs for the three families were 0.86, 1.62, and 1.24 mg/h
respectively.
Nagy et al. [77] described a strategy to purify protected carbohydrates via alternate-pump
recycling HPLC. In such a protocol, recycling of the target compounds was carried out
between two columns avoiding flowing back into the solvent pump. The developed
alternate-pump system gave the advantage of avoiding peak broadening associated with
the direct-pump system due to pumping the analyte back into the solvent pump as well as
the reversal of the concentration profile of the inlet stream. This protocol was easily
transferred to the preparative and semi-preparative scale for purification of larger
amounts with high throughputs. The recycling preparative HPLC approach has been also
applied for separation and purification of curcumin from curcuminoids in Curcuma
Longa L. In this method, the target compounds returned to the column after being in the
detector several times to attain a greater purity. The profits of this strategy include
enhancement of the resolution, higher product purity, and recovery yield and also
decreasing the solvent consumption and costs. The recycled preparative HPLC increased
the purity to about 99.5% [78]. Another approach has been implemented by Shen and
colleagues [79] for greening preparative HPLC by replacing the highly toxic solvents
with safer ones. Ethanol, acetone, and ethyl acetate are favorable greener alternatives to
methanol, acetonitrile, and tetrahydrofuran. These solvents are less toxic, cheaper, work
with the same or even better efficiency than traditional solvents for HPLC. Five closely-
related terpene trilactones from Ginkgo biloba extract have been separated adopting this
approach by gradient elution with water and a quaternary solvent of water-acetone-
ethanol-ethyl acetate (22.2:52.65:8.6:16.55, v/v).
Along with this approach for the replacement of hazardous solvents with safer ones,
hydrophilic interaction liquid chromatography (HILIC) appears to be a promising tool to

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perform purification of pharmaceuticals on the preparative scale. HILIC is a recent LC

technique that found rapid growth in popularity for separation of hydrophilic analytes.
HILIC employed a polar stationary phase (silica, alumina, amide) and an organic solvent-
rich mobile phase with 5-40% aqueous buffer content. The most applicable organic
solvent for HILIC techniques is acetonitrile, yet some applications utilized the benchmark
organic solvent ethanol. Recently, Jaffuel and colleagues [80] studied at-column dilution
to achieve improved resolution of plant root aqueous extract by preparative HILIC using
acetonitrile/aqueous buffer as the mobile. The encouraging results of such study inspired
extending the investigations while using ethanol as a greener solvent in order to develop
greener preparative HILIC methods for pharmaceutical purification.

Conclusions
Purification of API plays a key role during the manufacturing of pharmaceuticals. For
this aim, chromatographic techniques occupy a special place in the array of the available
purification techniques. CCC, SFC, and HPLC are valuable chromatographic techniques
for purification of pharmaceuticals on a large scale with better loadability and high
throughput. HPTLC finds great applications during synthesis of API, drug discovery, and
during the scale-up process prior to preparative chromatographic purification. As well,
GC also has wide applicability for residual solvents and impurities detection in API.
Despite SFC, CCC, and GC are considered inherently green chromatographic techniques,
many additional approaches have been adopted for attaining greater greenness. Analytical
Chemists have made great efforts for greening HPLC and TLC techniques via solvent
recycling and/or using greener solvents. These efforts combined all these
chromatographic techniques as green purification techniques for pharmaceuticals that
satisfy the society needs for green chemical methodologies that conserve the environment
and human safety.
Dedication
Rania El-Shaheny dedicates her efforts in preparation of this chapter with deep
appreciation and respect to the kind soul of the late great Egyptian author: Dr. Ahmed
Khaled Tawfiq who will remain immortal in the minds of the entire generation“until the
stars burn ....“.
Mahmoud El-Maghrabey wants to dedicate his contribution in this chapter to the soul of
his father who was the spark to every success he made.

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List of abbreviation
Abbreviation Meaning
API Active pharmaceutical ingredient
BP British Pharmacopoeia
CCC Countercurrent chromatography
CPE Cloud point extraction
2D Two-dimensional
GC Gas chromatography
HILIC-HPTLC Hydrophilic interaction liquid chromatography-high-
performance thin layer chromatography
HPLC High-performance liquid chromatography
HPTLC High-performance thin layer chromatography
IL Ionic liquid
LC Liquid chromatography
MAE Microwave-assisted extraction
ME Membrane extraction
MS Mass spectrometry
NP Normal phase
Ph. Eur. European Pharmacopoeia
PLE Pressurized-liquid extraction
PTV Programmed temperature vaporizer
RP Reversed-phase
RP-HPTLC Reversed-phase high-performance thin layer chromatography
SFC Supercritical fluid chromatography
SFE Supercritical fluid extraction
SPE Solid-phase extraction
SO-HPTLC Salting-out high-performance thin layer chromatography
SO-TLC Salting-out thin layer chromatography
SWE Supercritical water extraction
TD Thermal desorption
TGA Thermogravimetry
TLC Thin layer chromatography
UAE Ultrasound-assisted extraction
UPTLC Ultra-performance thin layer chromatography
USP United States Pharmacopoeia
VPE Vapor phase extraction

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