**3. Techniques of extraction and purification**

Analytical methods usually contain several steps, such as sampling, sample preparation, isolation, and quantification. Remarkably sample preparation just recently is concentration as an important analytical step. According to Wen *et al*. [39, 40], the main objective of sample preparation are removal of interferences, and preconcentration of the analytes that is considered a bottleneck of analytical processes. In this chapter, we will provide the current state of the art in sample preparation for analyzing alkaloids in herbal matrices, focusing on extraction, clean-up steps and purification.

### **3.1 Extraction**

## *3.1.1 Ultrasound assisted extraction (UAE)*

Ultrasound Assisted Extraction (UAE) technique is based on the using of acoustic waves in the kilohertz range spreading in liquid medium. These waves created by the ultrasound produces regions of compression and rarefaction in the molecules. Then, the cavitation bubbles are formed and collapse giving rise to smaller bubbles that could act as new cavitation nuclei or simply get dissolved. When the bubbles collapse at the surface of the herbal material, a shockwave having very high temperature and pressure is induced, resulting in plant cell disruption which enhances both the mass transfer of alkaloids into solution and the solvent penetration [41]. In addition, the swelling of plant materials can be enhanced by ultrasound, leading to improving of solvent penetration which increases the extraction yield [42]. The UAE procedure is optimized with regard to extraction solvent, temperature and liquid to solid ratio for the plant sample [41]. UAE has some advantageous properties including high extraction efficiency, good reproducibility, low solvent consumption, low cost and environmental friendliness. However, the major disadvantage of UAE is generating heat, leading to the degradation of thermo labile products and racemization of chiral compounds [43]. To avoid such types of drawback, extraction is carried on under an ice bath to reduce the temperature [44]. **Table 1** lists examples of protocols that were developed using MAE from various plants.

#### *3.1.2 Microwave assisted extraction (MAE)*

The MAE technique uses the electromagnetic radiations with a frequency range of 0.3–300 GHz, that stimulates ion migration and dipole rotation leading to the heating of dielectric materials and the penetration of extraction solvent into the matrix [49]. The released thermal energy is increased gradually with higher dielectric constant, so the effectiveness of MAE is depended on the dielectric properties of both extraction solvent and sample matrix [50]. Therefore, only specific solvents which have high dipole moment as water, methanol and ethanol can be used for extraction solvent in MAE and the moisture of plant sample is an important factor in the extraction efficiency [51]. Basically, higher water content matrices will be expected to give higher extraction yields. Water contained in plant matrices absorbs microwave radiation creating pockets of localized heating in the sample. This heat promotes the plant cell walls rupture which enhances the release of alkaloids into the solvent and the increase of extraction yield [50]. In addition to having a high dielectric constant, the extraction solvents must have a high dissipation factor to reduce the potential of localized sample overheating resulting degradation of alkaloids in plants [43]. Therefore, organic solvent-water mixtures, polar

**47**

*Chiral Alkaloid Analysis*

Macleaya cordata

Catha edulis

Ipomoea genera

Carica papaya

**Table 1.**

*DOI: http://dx.doi.org/10.5772/intechopen.96009*

Protopine Allocryptopine Sanguinarine Chelerythrine Dihydrochelerythrine Dihydrosanguinarine

> Norephedrine Cathine Cathinone

Ergot alkaloid (ergine, ergometrine, lysergic acid α-hydroxyethylamide) Penniclavine Chanoclavine Lysergol

Carpaine Pseudocarpaine Dehydrocarpaine I Dehydrocarpaine II

*Extraction conditions from various plants using UAE.*

**Plant Compounds Solvent Solvent:** 

1-hexyl-3 methylimidazolium tetrafluoroborate ([C6MIM][BF4]) aqueous solution

organic solvents and water are usually used as extraction solvent. Moreover, other parameters relating to extraction performance as sample size, sonication power, solid to liquid ratio, extraction time and microwave power should be modified for MAE procedure optimization [34]. The main advantages of MAE are the low solvent consumption, the ability to extract many samples simultaneously and the short extraction time [43, 52]. The major drawbacks of MAE are nonhomogeneous heating distribution and overheating of extract which may cause racemization or thermal degradation of chiral alkaloids [44]. **Table 2** lists examples of protocols that

The SFE process utilizes pressurized fluids (mainly CO2) as extraction solvents. In this technique, a fluid is heated and compressed to reach above critical point of it's creating the fluid having physicochemical properties of both liquid and gas states called supercritical fluid [58]. Specifically, supercritical fluid has a density

a diffusion coefficient that is intermediate between liquid and gas [59]. Therefore, supercritical fluid has higher transport capacity which facilitate to fluid diffusion through plant materials in comparison to traditional extraction solvents [43]. In addition, the density is related to polarity property of fluid which directly impact in solubility of compounds in extraction solvent. This parameter can be modified by controlling temperature and/or pressure so the flexibility and selectivity of the technique is enhanced, enabling selective extraction of different compounds from the plant matrix [60]. Carbon dioxide is commonly used in SFE because it has ideal properties including low critical temperature (31.3°C) and can be easily remove from extracts [51]. However, carbon dioxide is less effective in extraction of polar compounds from matrix because of its low polarity property. Aiming to extract

), a viscosity similar to gas (10−4 – 10−3 g/s.cm) and

**biomass (mL: g)**

**Temp (°C)**

0.1 N HCl 600: 1 — 45 [46]

70% methanol 100: 1 60 30 [47]

100% methanol 100: 7.5 — 20 [48]

500: 1 80 15 [45]

**Time duration (min)**

**Ref.**

were developed using MAE from various plants.

*3.1.3 Supercritical fluid extraction (SFE)*

similar to liquid (0.3–0.8 g/cm3


**Table 1.**

*Current Topics in Chirality - From Chemistry to Biology*

*3.1.1 Ultrasound assisted extraction (UAE)*

purification.

**3.1 Extraction**

MAE from various plants.

*3.1.2 Microwave assisted extraction (MAE)*

**3. Techniques of extraction and purification**

Analytical methods usually contain several steps, such as sampling, sample preparation, isolation, and quantification. Remarkably sample preparation just recently is concentration as an important analytical step. According to Wen *et al*. [39, 40], the main objective of sample preparation are removal of interferences, and preconcentration of the analytes that is considered a bottleneck of analytical processes. In this chapter, we will provide the current state of the art in sample preparation for analyzing alkaloids in herbal matrices, focusing on extraction, clean-up steps and

Ultrasound Assisted Extraction (UAE) technique is based on the using of acoustic waves in the kilohertz range spreading in liquid medium. These waves created by the ultrasound produces regions of compression and rarefaction in the molecules. Then, the cavitation bubbles are formed and collapse giving rise to smaller bubbles that could act as new cavitation nuclei or simply get dissolved. When the bubbles collapse at the surface of the herbal material, a shockwave having very high temperature and pressure is induced, resulting in plant cell disruption which enhances both the mass transfer of alkaloids into solution and the solvent penetration [41]. In addition, the swelling of plant materials can be enhanced by ultrasound, leading to improving of solvent penetration which increases the extraction yield [42]. The UAE procedure is optimized with regard to extraction solvent, temperature and liquid to solid ratio for the plant sample [41]. UAE has some advantageous properties including high extraction efficiency, good reproducibility, low solvent consumption, low cost and environmental friendliness. However, the major disadvantage of UAE is generating heat, leading to the degradation of thermo labile products and racemization of chiral compounds [43]. To avoid such types of drawback, extraction is carried on under an ice bath to reduce the temperature [44]. **Table 1** lists examples of protocols that were developed using

The MAE technique uses the electromagnetic radiations with a frequency range of 0.3–300 GHz, that stimulates ion migration and dipole rotation leading to the heating of dielectric materials and the penetration of extraction solvent into the matrix [49]. The released thermal energy is increased gradually with higher dielectric constant, so the effectiveness of MAE is depended on the dielectric properties of both extraction solvent and sample matrix [50]. Therefore, only specific solvents which have high dipole moment as water, methanol and ethanol can be used for extraction solvent in MAE and the moisture of plant sample is an important factor in the extraction efficiency [51]. Basically, higher water content matrices will be expected to give higher extraction yields. Water contained in plant matrices absorbs microwave radiation creating pockets of localized heating in the sample. This heat promotes the plant cell walls rupture which enhances the release of alkaloids into the solvent and the increase of extraction yield [50]. In addition to having a high dielectric constant, the extraction solvents must have a high dissipation factor to reduce the potential of localized sample overheating resulting degradation of alkaloids in plants [43]. Therefore, organic solvent-water mixtures, polar

**46**

*Extraction conditions from various plants using UAE.*

organic solvents and water are usually used as extraction solvent. Moreover, other parameters relating to extraction performance as sample size, sonication power, solid to liquid ratio, extraction time and microwave power should be modified for MAE procedure optimization [34]. The main advantages of MAE are the low solvent consumption, the ability to extract many samples simultaneously and the short extraction time [43, 52]. The major drawbacks of MAE are nonhomogeneous heating distribution and overheating of extract which may cause racemization or thermal degradation of chiral alkaloids [44]. **Table 2** lists examples of protocols that were developed using MAE from various plants.

## *3.1.3 Supercritical fluid extraction (SFE)*

The SFE process utilizes pressurized fluids (mainly CO2) as extraction solvents. In this technique, a fluid is heated and compressed to reach above critical point of it's creating the fluid having physicochemical properties of both liquid and gas states called supercritical fluid [58]. Specifically, supercritical fluid has a density similar to liquid (0.3–0.8 g/cm3 ), a viscosity similar to gas (10−4 – 10−3 g/s.cm) and a diffusion coefficient that is intermediate between liquid and gas [59]. Therefore, supercritical fluid has higher transport capacity which facilitate to fluid diffusion through plant materials in comparison to traditional extraction solvents [43]. In addition, the density is related to polarity property of fluid which directly impact in solubility of compounds in extraction solvent. This parameter can be modified by controlling temperature and/or pressure so the flexibility and selectivity of the technique is enhanced, enabling selective extraction of different compounds from the plant matrix [60]. Carbon dioxide is commonly used in SFE because it has ideal properties including low critical temperature (31.3°C) and can be easily remove from extracts [51]. However, carbon dioxide is less effective in extraction of polar compounds from matrix because of its low polarity property. Aiming to extract


#### **Table 2.**

*Extraction conditions from various plants using MAE.*

more polar alkaloids, the modifiers such as methanol, ethanol or water are added to extend the range of the solvating strength [43, 47]. For optimization of SFE procedure, these parameters such as pressure, temperature, modifier, flow of carbon dioxide and modifier [51]. Besides, the extraction time also effects on extraction yield, since an inadequate extraction time can result in incomplete extraction, while too long extraction time can cause the degradation of compounds. The major drawback of SFE are the complexity of system configuration and the requirement for a personal training program to operate the instrument [61].

#### *3.1.4 Pressurized solvent extraction (PSE)*

PSE process uses the pressurize solvents to enhance transport capacity of solvents and mass transfer rates which leads to improve extraction performance. In this technique, extraction solvents are heated at/or above the solvent's boiling points to decrease viscosity while keeping its in liquid state thanks to an elevated pressure [62]. Therefore, the extraction process is enhanced kinetic which leads to decreasing both the extraction time and solvent consumption. Similar to SFE, these parameters such as solvents nature, temperature and pressure should be modified to optimize PSE procedure [51]. Logically, higher temperature would be expected to give higher alkaloid extraction yields. However, excessive temperature may cause

**49**

*Chiral Alkaloid Analysis*

**3.2 Purification**

*DOI: http://dx.doi.org/10.5772/intechopen.96009*

for a professional training to operate instrument [43].

Extraction (SPME) have also been developed.

*3.2.1 Liquid–liquid extraction (LLE)*

*3.2.2 Solid phase extraction (SPE)*

analytes and evaporated to enrich sample [64].

degradation and racemization of chiral alkaloids. The main advantages of PSE are utilizing an extensive range of solvents (except strong acids/bases), low solvent consumption, short extraction time, automated instruments and performing an oxygen- and light – free extraction condition. Besides, the major drawbacks of PSE are similar to SFE such as using expensive laboratory equipment and requirement

Due to the alkaloids usually exist in plants at low concentration and the complication of plant matrices, samples should be purified and enriched which facilitate to identify and/or quantify process right after extraction step. Liquid–liquid extraction (LLE) and solid phase extraction (SLE) are popular clean-up methods utilized for sample preparation of alkaloids. Moreover, other techniques based on LLE and SPE methods, such as Liquid Membrane Extraction (LME) and Solid-Phase Micro

Liquid–Liquid Extraction (LLE) is the most simple and traditional clean-up method. LLE method is based on the relative solubility of compounds between two immiscible solvents. The alkaloids have polarity varying between pH, so the solubility of alkaloids in specific solvent are also affected by pH [34]. In the acid solutions (pH is lower than pKa of alkaloids), the analytes are protonated which leads to better water solubility so this aqueous phase can be washed with less polar organic solvents such as ethyl acetate, n-hexane and diethyl ether to eliminate hydrophobic interferences. After that, this aqueous layer is alkalinized which leads to the alkaloids becoming non-polarity and can be extracted by organic solvents to eliminate hydrophilic interferences [63]. For enrichment, the organic solvents layer can be collected, vaporized and reconstituted into new solvents which is suitable for analytical instrument. The main disadvantages of this method are requirement for

repetitive extraction causing time consuming and solvent wasting [51].

To overcome the drawback of LLE method, solid phase extraction (SPE) has been developed and applied in sample preparation since the 1970s. In this method, extract is loaded onto a sorbent phase which will retain alkaloids. Then, interferences in extract are washed away and the analytes is eluted by suitable solvents [64]. In fact, cartridge is the most popular SPE type due to its convenience. The SPE procedure have five step including conditioning, loading, washing and elution step. Several factors can affect to the extraction efficiency such as concentration of analytes in solvent, loading solvent nature, sorbent types, particle size, volume used for loading – washing – eluting, flow rate and elution solvent. Each factor has specified role which depends on the affinity of analytes and solid phase. Because the chemical structures of alkaloids always have secondary or tertiary amine groups, the strong cation exchange (SCX) sorbents are an ideal choice for sample preparation. When using these sorbents, washing solvent will be water and organic solvent to eliminate both hydrophilic and hydrophobic from plant matrices. After that, alkaloids will be deprotonated for elution by alkalized solvent which has pH at 2 units above pKa of

If the analytes are unstable in strong alkaline solutions, the weak cation exchange (WCX) will be use instead. The WCX sorbent has carboxylic acid as

#### *Chiral Alkaloid Analysis DOI: http://dx.doi.org/10.5772/intechopen.96009*

degradation and racemization of chiral alkaloids. The main advantages of PSE are utilizing an extensive range of solvents (except strong acids/bases), low solvent consumption, short extraction time, automated instruments and performing an oxygen- and light – free extraction condition. Besides, the major drawbacks of PSE are similar to SFE such as using expensive laboratory equipment and requirement for a professional training to operate instrument [43].

## **3.2 Purification**

*Current Topics in Chirality - From Chemistry to Biology*

Vasicine, Harmalin Harmine

Sinoacutine Palmatine Isocorydine L-tetrahydro palmatine

Dauricine Isoliensinine Neferine Nuciferine

Protopine Palmatine Allocryptopine Jatrorrhizine Tetrahydro palmatine Corypalmine Bicuculline

Bianfugedine, Menisporphine, 6-O-demethyl menisporphine Bianfugecine Dauriporphine Dauriporphinoline

Lotus plumule Liensinine

Peganum harmala

Stephania sinica

Corydalis decumbens

Menispermum dauricum

**Table 2.**

**Plant Compounds Solvent Solvent:** 

**biomass (mL: g)**

80% ethanol

65% ethanol

65% methanol

90% methanol

> 70% ethanol

**Tem (°C)**

**Time duration**

30: 1 80 8 min 600 [53]

24: 1 60 90 s 150 [54]

— — 4 min 200 [55]

20: 1 40 5 min — [56]

20: 1 60 11 min — [57]

**Micro wave power (W)**

**Ref.**

more polar alkaloids, the modifiers such as methanol, ethanol or water are added to extend the range of the solvating strength [43, 47]. For optimization of SFE procedure, these parameters such as pressure, temperature, modifier, flow of carbon dioxide and modifier [51]. Besides, the extraction time also effects on extraction yield, since an inadequate extraction time can result in incomplete extraction, while too long extraction time can cause the degradation of compounds. The major drawback of SFE are the complexity of system configuration and the requirement

PSE process uses the pressurize solvents to enhance transport capacity of solvents and mass transfer rates which leads to improve extraction performance. In this technique, extraction solvents are heated at/or above the solvent's boiling points to decrease viscosity while keeping its in liquid state thanks to an elevated pressure [62]. Therefore, the extraction process is enhanced kinetic which leads to decreasing both the extraction time and solvent consumption. Similar to SFE, these parameters such as solvents nature, temperature and pressure should be modified to optimize PSE procedure [51]. Logically, higher temperature would be expected to give higher alkaloid extraction yields. However, excessive temperature may cause

for a personal training program to operate the instrument [61].

*3.1.4 Pressurized solvent extraction (PSE)*

*Extraction conditions from various plants using MAE.*

**48**

Due to the alkaloids usually exist in plants at low concentration and the complication of plant matrices, samples should be purified and enriched which facilitate to identify and/or quantify process right after extraction step. Liquid–liquid extraction (LLE) and solid phase extraction (SLE) are popular clean-up methods utilized for sample preparation of alkaloids. Moreover, other techniques based on LLE and SPE methods, such as Liquid Membrane Extraction (LME) and Solid-Phase Micro Extraction (SPME) have also been developed.

## *3.2.1 Liquid–liquid extraction (LLE)*

Liquid–Liquid Extraction (LLE) is the most simple and traditional clean-up method. LLE method is based on the relative solubility of compounds between two immiscible solvents. The alkaloids have polarity varying between pH, so the solubility of alkaloids in specific solvent are also affected by pH [34]. In the acid solutions (pH is lower than pKa of alkaloids), the analytes are protonated which leads to better water solubility so this aqueous phase can be washed with less polar organic solvents such as ethyl acetate, n-hexane and diethyl ether to eliminate hydrophobic interferences. After that, this aqueous layer is alkalinized which leads to the alkaloids becoming non-polarity and can be extracted by organic solvents to eliminate hydrophilic interferences [63]. For enrichment, the organic solvents layer can be collected, vaporized and reconstituted into new solvents which is suitable for analytical instrument. The main disadvantages of this method are requirement for repetitive extraction causing time consuming and solvent wasting [51].
