HO CHI MINH NATIONAL UNIVERSITY

UNIVERSITY OF TECHNOLOGY

OFFICE FOR INTERNATIONAL STUDY PROGRAMS (OISP)

FACULTY OF CHEMICAL ENGINEERING

DEPARTMENT OF FOOD TECHNOLOGY

EXTRACTION METHODS AND

ANALYTICAL PROCEDURES FOR
ANTHOCYANIDINS

Instructor: Prof. Nguyễn Thị Lan Phi

Students: Đỗ Thành Nhân-1852626

Trần Hiến-1752195
NguyễnThị Minh Thu-1752313

Class: CC01
 School year: 2020 – 2021

Table of contents
CHAPTER 1.................................................................................................................................................................. 4
I. Anthocyanin characteristics.............................................................................................................................5
1. Definition........................................................................................................................................................5
2. Colors.............................................................................................................................................................5
II. Potential uses of anthocyanin pigments...........................................................................................................6
1. Antioxidants...................................................................................................................................................6
2. Relationship between diseases and Anothocynains.....................................................................................7
3. Mechanisms of action in disease prevention..............................................................................................10
Chapter 2..................................................................................................................................................................... 10
I. Sample preparation.........................................................................................................................................10
II. Anthocyanin extraction methods....................................................................................................................11
1. Anthocyanin solvent extraction..................................................................................................................11
2. Solid phase extraction.................................................................................................................................13
3. Pressurized fluid extraction........................................................................................................................15
4. Microwave assisted extraction....................................................................................................................18
5. Ultrasound assisted extraction....................................................................................................................19
6. ITV standard extraction method................................................................................................................19
7. AWRI based extraction method.................................................................................................................20
III. Analytical methods..........................................................................................................................................20
1. Liquid chromatography..............................................................................................................................20
2. Gas chromatography...................................................................................................................................25
3. 1H NMR spectroscopy.................................................................................................................................25
4. 13C NMR spectroscopy...............................................................................................................................26
5. Diode array detection systems (DAD)........................................................................................................26
6. Ultraviolet–visible spectroscopy.................................................................................................................26
7. Mass Spectrometry......................................................................................................................................26
8. Hydrolysis techniques..................................................................................................................................28
Chapter 3..................................................................................................................................................................... 28
Chapter 4..................................................................................................................................................................... 31
References.................................................................................................................................................................... 31
 List of figures
Figure 1: Anthocyanin structure.............................................................................................................3
Figure 2: Anothocyanin color change with pH levels............................................................................3
Figure 3:Solid phase extraction using macroporous resin...................................................................11
Figure 4: Pressurized solvent extraction method.................................................................................13
Figure 5: Microwave assisted extraction method.................................................................................16
Figure 6: Ultrasound assisted extraction method.................................................................................16
Figure 7: ITV extraction method..........................................................................................................17
Figure 8: Chemical structure of the main types of flavonoids.............................................................17
Figure 9: Structure of the flavylic cation and structure of the anthocyanidin cyanidin.......................18
Figure 10: Structure of anthocyanin cyanidin 3-glucoside..................................................................18
Figure 11: Possible structural transformations of anthocyanins in aqueous medium..........................19
Figure 12: HPLC method.....................................................................................................................20
Figure 13: Gas chromatography method..............................................................................................22
Figure 14: Mass spectrometry method.................................................................................................23
Figure 15: Working principle of MS....................................................................................................24
Figure 16: Sequential hydrolysis of typical grape anthocyanin...........................................................25

List of tables
Table 1: Anthocyanin color absorbtion..................................................................................................4
Table 2: Health benefits of anthocyanins...............................................................................................7
Table 3: The advantages and limitations of extraction procedures......................................................10
Table 4: Selected application of solid phase extraction.......................................................................11
Table 5: Selected examples of countercurrent chromatographic method............................................14
 CHAPTER 1
INTRODUCTION
I. Anthocyanin characteristics
1. Definition
Anthocyanin is an outstanding organic compound derived from nature. It belongs to the
Flavanoid group with extremely strong antioxidant capabilities. Luckily, anthocyanin can be
found easily in many different foods. The most prominent are the red, purple, and purple
vegetables and fruits.
The anthocyanins are all amphoteric forming salts with either acids or bases, anthocyanins
occur in plants as salts indicated by the positive charges.

Figure 1: Anthocyanin structure

2. Colors
 Figure 2: Anothocyanin color change with pH levels
Absorb light at about 500 nm, are the basis of the bright red, blue and purple color of fruits.
Every color except green has been observed (either natural or synthetic), depending on
aspects such as a kind of substituent present in the B-ring, the local pH, the state of
aggregation of the anthocyanins, complexation by organic molecules. Every color except
green has been observed (either natural or synthetic), depending on aspects such as a kind of
substituent present in the B-ring, the local pH, the state of aggregation of the anthocyanins,
complexation by organic molecules.

Table 1: Anthocyanin color absorbtion

The conjugated bonds in their structures (light-conjugated double bonds carrying a positive
charge), which absorb light at about 500 nm, are the basis of the bright red, blue and purple
color of fruits. Every color except green has been observed (either natural or synthetic),
depending on aspects such as a kind of substituent present in the B-ring, the local pH, the
state of aggregation of the anthocyanins, complexation by organic molecules

II. Potential uses of anthocyanin pigments

Anthocyanins extracted from plants have been used as food additives. Food additive, E163, is
one of the commercial additives derived from fruit anthocyanin such as grape skin. It is a
purple food additive for use in producing purple-colored jam, confectionaries, and beverages.
It’s currently known that synthetic food dyes attracted public concern regarding safety and
the adverse effect on human health, particularly neurological functions and behavioral
effects. 
 Another point is that the use of anthocyanin-based colorants in yogurt drink and some mixed
fruit juice is being more common. Moreover, acylated anthocyanins are food colorants used
in the food industry due to their high stability over nonacylated anthocyanins. Therefore, a
high level of nonacylated anthocyanins are produced from certain fruits, such as elderberry
and barberry, at relatively low cost. These commodities have potential as colorants for use in
the food industry.
1. Antioxidants
The glycosylated B-ring structure of anthocyanin contributes to the high antioxidant activity,
where ortho-hydroxylation and methoxylation substantially increase the antioxidant activity.
What’s more, anthocyanidin has a higher ORAC value than anthocyanin. One of the possible
reasons is anthocyanin aglycone is very unstable and highly reactive. Acylation of
anthocyanin with phenolic acid has a significant increase in antioxidant activity .Diacylation
of the anthocyanin markedly increases the antioxidant activity but 5-glycosylation leads to a
reduction in the activity.
Anothocynains have many other therapeutic effects in addition to their antioxidant activities.
As an active pharmaceutical ingredient, anthocyanin pigment, such as delphinidin, has been
patented for several therapeutic effects. Delphinidin is well-known for combating melanoma
cells , as well as antimicrobial effects, such as curing Staphylococcus aureus infection . It has
also been used as the source of antiphlogistic or immunosuppressive active ingredients.
On the other hand, a study demonstrates that cyanidin and cyanidin-3-glucoside have the
highest inhibitory effect on copper (II)-induced low-density lipoprotein (LDL) oxidation
compared with the other phenolic acids, anthocyanins, and anthocyanin aglycones, whereas
delphinidin has intermediate efficacy.

2. Relationship between diseases and Anothocynains

Angiogensis and development of diseases
Endothelial cells are the main cells involved in the angiogenesis process. Disturbances in
physiologic angiogenesis can contribute to various human diseases, including CVDs, cancer,
and diabetic complications such as diabetic retinopathy and nephropathy. Normal
angiogenesis depends on the intricate balance between angiogenic (VEGF, FGF2-fibroblast
growth factor, TGF-β-transforming growth factor, and angiopoietin) and antiangiogenic
(angiostatin, endostatin, and thrombospondins) factors. Anthocyanin-rich purple corn extract
attenuates endothelial expression of VEGF and hypoxia inducible factor (HIF)-1α, as well as
to induce endothelial marker of platelet endothelial cell adhesion molecule-1 and integrin β3
induced by high glucose condition in human renal mesangial and endothelial cells.
Cardiovascular health
Epidemiological studies show the relationships between anthocyanin-rich foods and CVDs,
as well as the relationship between total anthocyanin intake and risk of developing these
cardiovascular-related diseases.
Anthocyanins also demonstrate in vitro anti-thrombotic effect. To be more specific, the anti-
thrombotic effect is revealed by another study that anthocyanin-containing maize seed (20%
seed in the diet) fed rats for eight weeks are less susceptible to ischemia-reperfusion injury
and reduction of infarct size with increased myocardial antioxidant enzyme. Apart from that,
other experts ( Bell and Gochenaur) state that anthocyanin-rich extracts of chokeberry and
bilberry, but not elderberry, possess vasorelaxation properties. 
In terms of Biomarkers and mechanism for this. Oxidative damage to a cardiovascular
system is typically caused by ROS and RNS. During the development of CVD, oxidative
stress causes vascular inflammation. Vascular inflammation alters the levels of cellular total
cholesterol (TC), LDL, high-density lipoprotein (HDL), and very low-density lipoprotein
(VLDL), as well as plasma malonaldehyde and other plasma enzymes (SOD, catalase, and
glutathione peroxidase) levels.
As a result, it obtained from a double-blind clinical trial of 150 subjects aged 40–65 years old
show that consumption of anthocyanins (total intake of 320 mg anthocyanins per day) for
24 weeks significantly reduced the serum high sensitivity C-reactive protein (−21.6%),
soluble vascular cell adhesion molecule-1 (−12.3%), and plasma IL-1β (−12.8%) compared
to the placebo (−2.5%, 0.4%, and −1.3%, respectively).
Anticancer
Anthocyanins have been extensively studied for their anticancer properties, as well as
antiangiogenesis, based on in vitro and cell culture studies, and animal models. Angiogenesis
is the key for cancer development, where it is an important step in the transition of tumors
from a benign state to a malignant one. In order to prevent cancer, antiangiogenesis is the
process that prevents formation of new blood vessels that supply oxygen to the tumor cells
In term of how they come from, anthocyanins have been extracted and isolated from different
plant sources for investigating their anticancer ability on esophagus, colon, breast, liver,
hematological, and prostate cancers. It’s noticed that the evidence from a previous study
shows that 5% whole freeze-dried black raspberries and the anthocyanin-rich fraction
supplemented to N-nitrosomethylbenzylamine-induced F344 rats have chemopreventive
potential, where the treatment groups inhibit cell proliferation, inflammation, angiogenesis,
and induce apoptosis in both preneoplastic and papillomatous esophageal tissues. Thus
anthocyanins have chemoprophylaxis potential.
In another experiment, supplementation of anthocyanin-rich extracts of bilberry, chokeberry,
and grape (containing 3.85 g anthocyanins per kg diet) for 14 weeks significantly reduced
azoxymethane-induced aberrant crypt foci by 26–29% in 3–4 weekold male-specific
pathogen-free F344 rats. This reduction is associated with reduced cell proliferation and
decreased expression of the COX-2 gene. The result also shows that the urinary 8-OHdG
levels were similar among rats fed with different diets.
In another study, black rice anthocyanins suppress metastasis in breast cancer cells by
targeting the mitogen-activated protein kinase pathway. The anthocyanins inhibited
migration and invasion of MDA-MB-453 cells (HER2+), suppressed activation of rapidly
accelerated fibrosarcoma, mitogen-activated protein kinase (MEK), and c-Jun N-terminal
kinase (JNK), as well as downregulated secretion of matrix metalloproteinase 2 (MMP2) and
MMP9. The study suggests that black rice anthocyanins suppress metastasis in breast cancer
cells by targeting the RAS/RAF/MAPK (retrovirus-associated DNA sequences/rapidly
accelerated fibrosarcoma/mitogen-activated protein kinase) pathway. Thus, it may be useful
to treat patients at an advanced cancer stage.
Antidiabetes
The antidiabetic effect of anthocyanins from plants has been widely studied. Anthocyanin-
rich Cornus fruits have been used in traditional Chinese prescription medicines to treat
diabetes. Primary bioactive components reported in Cornus fruits are the glycosides of
cyanidin, delphinidin, and pelargonidin. Another study demonstrates that pelargonidin and
pelargonidin-3-galactoside caused a 1.4-fold increase in insulin secretion at 4 mM glucose
concentration representative of the normal glucose level in human. The ability of the
anthocyanins to induce insulin secretion is in the increasing order of pelargonidin-3-
galactoside, cyanidin-3-glucoside, and delphinidin-3-glucoside. This finding demonstrates
that the number of hydroxyl groups on the B-ring of anthocyanins plays a crucial role in their
ability to secrete insulin. Nevertheless, cyanidin, delphinidin, pelargonidin, malvidin, and
petunidin do not potentiate significant insulin secretion.
It has been reported that a reduction in AMPK activity leads to diabetic nephropathy, which
is associated with increased oxidative stress and lipid accumulation. Supplementation of
anthocyanin-rich Seoritae extract restores AMPK activity, activates target molecules such as
ACC, sterol regulatory element-binding protein 1, and PPAR, and suppresses intrarenal lipid
accumulation in kidney tissue. However, the authors did not examine specific contributions
of the bioactive compounds in the Seoritae extract to the observed effects and the amounts of
these compounds incorporated into the kidney. It is unsure if the target of anthocyanins is
only AMPK or adiponectin.
Aniti-obesity effect
Anthocyanidin and anthocyanin pigments possess anti-obesity properties. Based on a
previous study, obese mice fed a diet rich in cyanidin-3-glucoside from purple corn for
12 weeks have reduced body weight, as well as decreases in white and brown adipose tissue
weights. The study demonstrates that hyperglycemia, hyperinsulinemia, hyperleptinemia, and
an increase in the tumor necrosis factor (TNF-a) mRNA level occurred in the obese rats are
normalized when treated with purple corn diet. The purple corn also suppresses mRNA levels
of enzymes involved in fatty acid and triacylglycerol synthesis and lowered sterol regulatory
element binding protein-1 mRNA level in the white adipose tissue.
Obesity is strongly associated with adipocyte dysfunction. Therefore, regulation of protein
secretion from adipocyte or the adipocyte-specific gene expression is one of the most
important targets for prevention of obesity. Tsuda and his research team further investigated
the potency of anthocyanins, particularly cyanidin and cyanidin-3-glucoside on isolated rat
adipocytes for anti-obesity effect. He demonstrates that the adipocytes treated with
anthocyanins have increased adiponectin and leptin secretions and upregulated adipocyte-
specific gene expression without activation of PPARγ in the isolated rat adipocytes 
Antimicrobial
Polyphenolic compounds including anthocyanins possess antimicrobial activity against a
wide range of microorganisms, especially in inhibiting the growth of food-borne pathogens.
Anthocyanins exhibit antimicrobial activity through several mechanisms, such as induced
cell damage by destroying the cell wall, membrane, and intercellular matrix.
Based on a previous study, maqui berry extracts had antibacterial activity with the highest
sensitivity to Aeromonas hydrophilia and Listeria innocua. These bacteria are commonly
associated with refrigerated foods as indicators of pathogenic microorganisms or as spoilage
microorganisms. Côté et al. report that cranberry extract had antibacterial activity
towards Enterococcus faecium resistant to vancomycin, Pseudomonas aeruginosa,
Staphylococcus aureus, and Escherichia coli.
Anthocyanin-rich extracts, such as blueberry, raspberry, blackcurrant, and strawberry
extracts, inhibit Gram-negative bacteria but not Gram-positive bacteria. This variation may
be due to the different structures of cell wall between Gram-negative and Gram-positive
bacteria, in which the outer membrane of Gram-negative bacteria acts as a preventive barrier
against hydrophobic compounds but not on hydrophilic compounds. These antimicrobial
activities of anthocyanin-containing extracts are possibly due to the multiple mechanisms and
synergistic effects of various phytochemicals in the extracts, including anthocyanins, weak
 organic acids, phenolic acids, and their mixtures of different chemical forms . Thus, the
antimicrobial effect of chemically complex compounds instead of solely anthocyanins should
be extensively analyzed. Also, anthocyanins in purple, red, and blue-colored fruits and
vegetables are the main bioactives in preventing microbial infection by several mechanisms.
Table 2: Health benefits of anthocyanins

Health benefits of anthocyanins

Visual Health Ani-obesity Antimicobial
-Improved -Improved -Inhibited
visual function weight gain gram-negative
in patients and lipid bacteria but
with normal profile on not on gram-
tension obese rats. positive
glaucoma. bacteria
-Prevented -Suppressed -Possessed
impairment of weight gain, antimicrobial
photoreceptor fat tissue gain activity
cell function and other through
during retinal metabolic damaging and
inflammation. disorders destroying the
-Increased -suppressed cell wall,
ocular blood body weight membrane and
flows but no gain and intercellular
significant improved matrix
changes on blood lipid
intraocular profile in
pressure high-fat diet
induced rats

3. Mechanisms of action in disease prevention

Anthocyanins are the good antioxidants for preventing or reducing the risk of disease.
Anthocyanins reduce the risk of several diseases that can be shown by direct and indirect
pathways. Direct pathway is that the colored compounds directly reduce the risk of several
chronic diseases through scavenging free radicals and thus reducing oxidative stress. The
indirect pathways involve downregulation of cell proliferation and apoptosis through
reduction of oxidative stress and lipid peroxidation. It is commonly known that anthocyanins
are the strong antioxidants that effectively scavenge free radicals. Anthocyanins reduce the
risk of CVD through improving blood lipid profile and biomarkers. A reduction in certain
blood biomarkers is known to prevent CVDs. Similar to many other phenolic compounds,
anthocyanins inhibit cancer cell proliferation via several pathways. One of the well-known
mechanisms of action is the downregulation of cyclooxygenase (COX) enzyme activity.
These enzymes catalyze the formation of leukotrienes, prostacyclins, prostaglandins (PGs),
and thromboxanes. Downregulation of COX enzymes, including COX-1 and COX-2,
reverses cell proliferation and thus reduces the risk of cancer. Anthocyanins also inhibited
tumor growth by blocking activation of the mitogen-activated protein kinase pathway.
Moreover, the most commonly known pathways are cytokine signaling pathways. The
analysis of structure-activity relationships among flavonoids suggests that 4-hydroxylations
at positions 5, 7, 31, and 41, together with a bond at C2–C3, and the B-ring attaching at the
C2 position, seem necessary for the highest expression of monocyte chemoattractant protein
1 (MCP-1).
 CHAPTER 2
EXTRACTION METHODS AND ANALYTICAL PROCEDURES

I. Sample preparation

Air drying

Milling

Grinding

Homogenization

Filtration Centrifugation

II. Anthocyanin extraction methods

1. Anthocyanin solvent extraction
The analysis of anthocyanins is complex as a result of their ability to undergo structural
transformations and complexation reactions. Anthocyanins may be part of complexes, may
occur in complex matrices, and may exist in a variety of protonated, deprotonated, hydrated,
and isomeric forms, and the relative proportion of these molecules is strongly dependent on
 pH. Acid dissociation constants are important physicochemical parameters that describe the
extent of ionization of functional groups as a function of pH; they are of vital importance in
the analysis of bioactive compounds as well as in the interpretation of their mechanism of
action. Their antiradical properties, i.e., the ability to react quickly and efficiently with
electron-deficient radicals, depend on the acidity of phenolic hydroxyl groups and on the
stability of the formed radical.
Typical procedures for isolation and characterization of pure anthocyanins consist of several
steps:
(i) extraction of the plant material, followed by a preliminary purification step,
(ii) fractionation of the mixture followed by isolation of pure pigments, and finally
(iii) characterization and identification of pure anthocyanins. Methods such as solid-phase
extraction, as shown later, use solid adsorbents to extract phytochemicals from liquid
matrixes such as juices. It is easy, rapid, and economical compared to solvent extraction.
However, SPE is perhaps more often used in sample cleanup, purification or pre-
concentration than in extraction because of the selectivity and saturation of the adsorbents.
Extraction is a very important stage in the isolation, identification, and use of anthocyanins.
The recovery of anthocyanins is commonly performed through a solvent-extraction
procedure and the solvent type, solvent concentration, liquid-to-solid ratio, temperature, and
time are important parameters to be optimized. The extracting solution should be slightly
acidic to maintain the flavylium cation form, which is red and stable in highly acidic
medium, but not so acidic to cause partial hydrolysis of the acyl moieties in acylated
anthocyanins. The structural diversity, together with the susceptibility of anthocyanins to
heat, pH, metal complexing, and copigmentation, complicates the protocols of extraction and
analysis from both plant material and biological fluid.
Anthocyanins are heat-sensitive, so high temperatures must be avoided during extraction and
concentration, i.e., <30◦C. Anthocyanins, like flavonoids in general, have aromatic rings
containing polar substituent groups and glucosil residues that altogether produce a polar
molecule. While flavonoid glycosides are more polar, aglycones are extracted with alcohols
or alcohol-water mixtures. Anthocyanins are extracted with cold acidified solvents under
mild conditions. The organic solvent is usually methanol, but many other solvents may be
used such as acetone, ethanol, or acetonitrile. This solvent system denatures the cell
membranes, simultaneously dissolving the anthocyanins and stabilizing them. The acid
employed is usually acetic acid or trifluoroacetic acid, whereas the organic solvent content
varies from 50% to 100% of the mixture. The use of mineral acid can lead to the loss of
attached acyl group. Sulfured water has also been used as extraction solvent in seeking a
reduction of the use of organic solvents as well as the cost of extraction.
Anthocyanins are normally extracted with methanol containing 0.5% trifluoracetic acid.
Black beans were also presoaked in water containing 0.5% TFA. In this case this was done to
improve the extraction yields of anthocyanins because direct methanolic extractions provide
very poor yield. The extraction was performed in a refrigerator at low temperatures to avoid
hydrolysis of potential acyl groups in the anthocyanin structure and degradation. After
extraction the extract was filtered, and themethanol was removed by evaporation under
reduced pressure at relatively low temperatures.
Anthocyanins are also extracted under cold conditions with methanol, if it contains 2–10%
formic acid. This process is very good for grape extraction despite the fact that the final
wateralcohol extract will include some non-polyphenolic substances such as sugars, amino
acids, proteins, and pigments. Thus, the extract must be purified. If appreciable amounts of
lipids, chlorophylls, or unwanted polyphenols are suspected to be present in anthocyanin-
containing extracts, they may be removed by washing with ethyl acetate, petroleum ether,
ethyl ether, or diethyl ether. A procedure used acetone as the extracting solvent followed by
separation of the aqueous phase by addition of chloroform, isolating and partially purifying
the pigments in this way. The aqueous portion contains the anthocyanin, phenolics, sugar,
organic acids, and other water-soluble compounds, whereas the choroform phase contains the
lipids, carotenoids, chorophyll pigments, and other nonpolar compounds. High recoveries of
anthocyanins are obtained requiring little concentration or further purification .
Column chromatography has been also employed for fractionation of phenolic extracts. This
method, though often labor intensive and solvent consuming, ensures that greater amounts of
fractions can be obtained for use in subsequent isolation and identification of pure
substances.
The composition of anthocaynins and polyphenols is highly dependent on the extraction
temperature,which reflects the conflicting actions of solubilization and analyte degradation.
Under cold extraction, color degradation was significantly lower and extraction times were
15-fold longer. A 50% methanol extract shows maximum recovery for anthocyanins bunga
kantan inflorescence. Eight different solvent mixtures containing acetone or methanol, pure
or combined with an acid, were tested for their efficiency for extraction of phenolic
compounds from strawberries belonging to five groups of polyphenols including
anthocyanins.
Solvent extraction offers good recovery of antioxidant phytochemicals from various samples.
The primary drawback of the traditional extraction procedure is that the obtained final
extracts often require subsequent concentration and cleanup prior to analysis. Furthermore,
when considering the extraction of bioactive compounds, which are unstable and
thermolabile and are found in low concentrations, traditional extraction techniqueswould not
be themost suitable option. However, the use of large amounts of organic solvents poses
health and safety risks and is environmentally unfriendly. There are many alternative
methods that either eliminate or reduce significantly the use of organic solvents, e.g.,
microwaveassisted extraction, supercritical fluid extraction, and pressurized liquid extraction,
whose use is increasingly popular in the extraction of antioxidant phytochemicals. Some of
these methods offer identical, if no better, extraction efficiency and cost effectiveness.
Table 3: The advantages and limitations of extraction procedures.
2. Solid phase extraction
Solid phase extraction was developed in the 1980s and has emerged as a powerful tool for
chemical isolation and purification. The goals of SPE are usually retention and elution of an
analyte from a sample, removal of contaminants and interfering substances, and sample
concentration. It is based on the same principle of affinity-based separation as liquid
chromatography and overcomes the limitations of liquid-liquid extraction. It requires
application of samples in a liquid state, a proper extraction being the first preparation step of
solid samples. Simple filtration or centrifugation and then a filtrate or supernatant is applied
to the SPE cartridge.

Figure 3:Solid phase extraction using macroporous resin

SPE on C18 cartridges or Sephadex is commonly used for the initial purification of the crude
anthocyanin extracts. Although they are available in normal phase, reverse-phase, and ion-
exchange modes, reversephase adsorbents are used as the main adsorption materials in SPE.
The anthocyanins are bound strongly to these adsorbents through their unsubstituted
hydroxyl groups and are separated from unrelated compounds by using a series of solvents or
increasing polarity.
Currently used methods for anthocyanin extraction are nonselective and result in solutions
with large amounts of undesirable products such as sugars, acids, amino acids, and proteins
that require removal. Crude extracts have been purified by removal of sugars, acids,
andwater-soluble compounds with C18 cartridges previously activated with methanol,
followed by water or 0.01% aqueous HCl or 3% formic acid. Partitioning of extracts with
ethylacetate has been shown to remove interferences prior to LC-MS analysis.
Table 4: Selected application of solid phase extraction
 Anthocyanins were recovered from diluted fruit juice or wine by elution from a C18 cartridge
with an aqueous eluent at a low pH. However, SPE was used to obtain anthocyaninrich
extracts from berry species and to obtain a pigment-rich fraction in anthocyanins from grape
skins as well as in the anthocyanin analysis of cranberry fruit and cranberry fruit products,
prior to the HPLC final determination. Shah and Chapman used mixed-mode cation
exchange SPE to purify anthocyanins from tulip extracts in 50:50 methanol:water with 0.1%
formic acid, and Ling et al. have used hydrophilic-lipophilic balanced SPE to extract
anthocyanins from human tissue homogenates. Automated off-line SPE can be used in
combination with LC-MS-MS to meet specific needs. Applications of SPE to anthocyanins
are compiled in Table 4.
3. Pressurized fluid extraction
Supercritical fluid extraction represents an interesting alternative technique to conventional
solid-liquid extraction with lower solvent consumption and lower working temperature. It is
a form of liquid extraction where the usual liquid solvent phase has been replaced by a
supercritical fluid; i.e., a substance that is above its critical point. Among a wide variety of
supercritical fluids, carbon dioxide is essentially the only convenient supercritical extraction
solvent used because of its comparatively low critical temperature and pressure. This method
provides an alternative to the pretreatment of the plant materials, replacing toxic organic
solvents.
The extraction of anthocyanins by using supercritical CO2 methods requires high pressures
and the presence of an organic co-solvent in high percentage due to the polarity of
anthocyanins. High concentrations of polar organic modifiers added to obtain the best
extraction yield lead, however, to reduced selectivity. Bleve et al. described amethod for the
purification of anthocyanins from grape skin extracts as liquidmatrix, by usingCO2 under
liquid and subcritical conditions. The solid residues generated from blueberries, cranberries,
and raspberries after pressing were extracted by conventional solvent extraction or by SC-
CO2 extraction. SFE of bioactive compounds from grape peel by using 6–7% ethanol as
modifier has been carried out.

Figure 4: Pressurized solvent extraction method

Fractionated high-pressure extraction is performed in order to obtain anthocyanin-rich
extracts. A first step with SC-CO2 followed by a second step with mixtures of CO2 and
ethanol were applied to cherries; the product derived from the second CO2-EtOH step
extraction exhibited activity against colon cancer. SCCO2 fluid extraction followed by
enhanced solvent extraction with CO2-EtOH-H2O mixtures allows obtaining
anthocyaninrich fractions in the second step, from elderberry pomace. Supercritical fluid
extraction, with 6–7% ethanol as modifier, was applied for the extraction of valuable
compounds, e.g., total anthocyanins, from grape peel.
Pressurized liquid extraction, also known under the trade names of accelerated solvent
extraction, pressurized fluid extraction, pressurized solvent extraction, or enhanced solvent
extraction, is a relatively new technology for extraction of phytochemicals under high
temperature and pressure and is partly derived from SFE. In PLE, pressure is applied to allow
the use as extraction solvents of liquids at temperatures greater than their normal boiling
point. The method was first described in 1995. The combined use of high pressures and
temperatures provides faster extraction processes that require small amounts of solvents.
There are two ways to perform PLE, either in the static or dynamic mode. In both cases,
under conditions of elevated pressure and temperature, the mass transfer rates are accelerated
according to Fick’s law of diffusion. Both commercially available and laboratory-assembled
PLE systems are used.
Therefore, extraction solvents, including water, that show low efficiency in extracting
phytochemicals at low temperatures may be much more efficient at elevated PLE
temperatures. The use of water as an extraction solvent in PLE is the so-called pressurized
hot water extraction. PHWE is also referred to as subcritical water extraction, superheated
water extraction, hightemperature water extraction, extraction using hot compressed water,
and extraction with water at elevated temperatures and pressures.Subcritical water appears to
be an excellent alternative to organic solvents to extract anthocyanins and other phenolics
from dried grape skin and possibly other grape byproducts, and from industrially generated
apple pomace.
Table 5: Selected examples of countercurrent chromatographic method.
PHWE is usually performed in dynamic mode with water flowing constantly through the
sample, but static extraction is also possible. Water is heated up to 200◦C and the change in
dielectric constant of the water with the temperature leads water to behave like an organic
solvent. For example, the dielectric constant of water at 200◦C is equal to 36, which is close
to that of methanol.
A dynamic subcritical fluid extraction system has been used for extraction of flavonoids from
food processing waste such as grape pomace. Anthocyanins have been extracted from red
grape pigments by PLE in a static model. A static bath reactor has also been used for the
extraction of anthocyanins from red onion. Pressurized hot water containing 5% ethanol was
 used to extract anthocyanins from red cabbage. Anthocyanins were extracted from red
pomace in acidified aqueous methanolic and aqueous ethanolic solvents and identified and
quantified in the extracts by HPLCMS and HPLC.
Residence time of the extracted solute must be minimized in order to prevent possible
degradation of anthocyanin moieties or their possible reaction with sugar and other products.
There is some evidence that side reactions generating antioxidant moietiesmay occur in
pressurized hotwater.
At first, the extraction effect dominates, but degradation effects soon take over, as occurs
with anthocyanins from red onion. Anthocyanins present in black carrot were extracted with
pressurized acidified water; anthocyanin degradation became significant above 100◦C. At the
present, no commercial instrument is available and, thus, the equipment has been self-built or
modified from other instruments. The difference from commercial instruments for SFE and
PLE or ASE is that PHWE equipment can tolerate temperatures over 300◦C. The high
temperature makes demands on the material of the extraction vessels and sealing rings. In
pressurized hot water extraction the most important parameter affecting extraction is the
temperature.
The other significant parameters are pressure, flow rate of the water, and extraction time. In
addition, the matrix needs to be taken into consideration, and choosing the proper analyte
collection system can increase selectivity of the extraction. Response surface methodology
was usually used to optimize the response values in the extraction of anthocyanins from
different sources.
4. Microwave assisted extraction
The microwave extraction procedure 50 g of berries were put into a kitchen blender and
mashed on a low pulsed level to break pulp and skin but leave the seeds intact. The mash was
then quantitatively transferred into a 120 mL plastic tube. 11 mL of 100% ethanol were
added to simulate wine extraction conditions and the tube was closed with a lid. After the
microwave was set to 700 W output and samples were microwaved for 2.5 min with short
interruptions for stirring every full minute and after completed extraction.

Figure 5: Microwave assisted extraction method

Juice was then decanted under low manual pressure with a potato ricer and centrifuged at
4200 RPM for 10 min. Extracts were transferred into 2 mL centrifuge tubes and centrifuged
with 13,000 RPM at 5oC for 10 min. Spectrophotometric absorbance was analyzed at 280,
 420, 520, and 620 nm. An aliquot of each extraction was stored at 20oC for further LC
analysis.
5. Ultrasound assisted extraction
Ultrasound assisted extraction was based on a method by Corrales: 50 g of berries were put
into a kitchen blender and mashed on a low pulsed level to homogenize pulp and skin but
leave the seeds intact. The mash was quantitatively transferred into a 120 mL plastic tube and
11 mL of 100% ethanol were added to simulate wine extraction conditions. The tubes were
placed into the sonicator for 60 min while stirring the mash every 10 min. Juice was then
decanted under low manual pressure with a potato ricer and centrifuged at 4200 RPM for 10
min. Extracts were transferred into 2 mL centrifuge tubes and centrifuged with 13,000 RPM
at 5oC for 10 min. Spectrophotometric absorbance was analyzed at 280, 420, 520, and 620
nm. An aliquot of each extraction was stored at 20oC for further LC analysis.

Figure 6: Ultrasound assisted extraction method

6. ITV standard extraction method
The ITV extraction method was adapted from the description of Cayla: 50 g of berries were
put into a kitchen blender and mashed on a low pulsed level to homogenize pulp and skin but
leave the seeds intact.
The mash was then quantitatively transferred into a 120 mL plastic tube and 15 mL of 96%
ethanol and 85 mL of 0.1% hydrochloric acid were added. The mixture was incubated for
one hour and shacked every 15 min. Juice was then decanted under manual pressure with a
potato ricer and centrifuged at 4200 RPM for 10 min. The supernatant was decanted and
analyzed at 420 and 620 nm. Every extract was then diluted 1:20 with the extraction solution.
Spectrophotometric absorbance of the diluted extracts was analyzed at 280 and 520 nm. An
aliquot of each undiluted extraction was stored at 20oC for further LC analysis.

Figure 7: ITV extraction method

 7. AWRI based extraction method
The extraction method for polyphenols used by the AWRI was modified from the
description: 20 g of berries were put into a kitchen blender and mashed on a low pulsed level
to homogenize pulp and skin but leave the seeds intact. 4 g of the mash were then
quantitatively transferred into a 50 mL plastic centrifuge tube. 4.4 mL of a 10 N hydrochloric
acid were added to 50% ethanol in water. 20 mL of this solution were then added to the
sample and agitated on a shaker table at 30 RPM for one hour at room temperature with more
intense manual shacking every 15 min. The samples were centrifuged at 4200 RPM for 10
min. Spectrophotometric absorbance of the supernatant was analyzed at 420 and 620 nm with
extraction solution as a reagent blank. Each sample was then diluted with 1% hydrochloric
acid and absorbance was analyzed at 280 and 520 nm with the 1% HCl as a reagent blank.
An aliquot of each undiluted extraction was stored at 20oC for further LC analysis.

III. Analytical methods

1. Liquid chromatography
Color is one of the most important quality attributes of a food, exerting an enormous
influence on its aesthetic value and serving as a basis for the acceptance of a wide variety of
food products by consumers. In natural products, most of the substances responsible for
coloring belong to the class of flavonoids.

Figure 8: Chemical structure of the main types of flavonoids

The flavonoids have a structure marked by the presence of a skeleton with 15 carbon atoms
in the form C -C -C, and are divided into classes depending on the oxidation state of the
central pyran ring. two Figure 8 shows the chemical structure of the main types of
flavonoids.
The classification of the type of flavonoid present in a plant extract is based initially on the
study of the solubility properties and staining reactions. This procedure is followed by
chromatographic analysis of the plant extract Flavonoids can be separated by
chromatographic procedures and the individual components
 Figure 9: Structure of the flavylic cation and structure of the anthocyanidin cyanidin
when possible, by comparison with standards. The two classes of flavonoids considered to be
the most important are flavonols and anthocyanidins. 4 Anthocyanidins have as their
fundamental structure the flavylic cation 5, represented in Figure 9a. Figure 9b shows an
example of anthocyanidin structure, known as cyanidin.
Pigments generally occur in the form of anthocyanins, 6 that are derived from
anthocyanidins. Anthocyanidins do not have glycoside groups and most have hydroxyls in
positions 3, 5 and 7. In anthocyanins, one or more of these hydroxyls are linked to sugars, the
most common of which are glucose, xylose, arabinose, rhamnose, galactose or disaccharides.
those sugars, to which phenolic acids may be attached, as P-coumaric, caffeic, phenyl and
vanillic. The sugar present in the anthocyanin molecules provides greater solubility and
stability to these pigments, when compared to anthocyanidins. Figure 10 is an example of
anthocyanin structure present in most vegetables, cyanidin 3-glucoside.

Figure 10: Structure of anthocyanin cyanidin 3-glucoside

With the same biosynthetic origin as the other national flavonoids anthocyanins are
structurally characterized by the presence of a skeleton containing 15 carbon atoms in the CC
-C form, however, unlike the other flavonoids, anthocyanins absorb strongly in the visible
region of the spectrum, giving an infinity of colors, depending on the medium of occurrence.
Due to its solubility in water, anthocyanins occur in the tissues of plants dissolved in the fluid
of the plant cell, which generally has a slightly acidic pH. The anthocyanins most commonly
found in fruits are mainly derived from six anthocyanidins: pelargonidin, cyanidin,
delfinidine, peonidine, pethidine and malvidin. The nomenclature of the pigments is derived
from the source from which they were first isolated. The differences between the various
anthocyanins are in the number of hydroxy groups in the molecule, the degree of methylation
of these groups, the nature and number of sugars attached to the molecule and the position of
these bonds, as well as the nature and number of aliphatic acids and / or aromatics linked to
the sugar in the anthocyanin molecule.
An outstanding feature of anthocyanins is the fact that in aqueous solutions, they have
different structures depending on the pH. In general, in an extremely acidic environment,
anthocyanins have an intense red color due to the predominance of the flavylic cation form.
For a medium larger than 2, a balance is observed between the flavylic cation and a structure
known as pseudobase carbinol. With an increase in pH, anthocyanins lose their color until
they become practically colorless at pH approximately, due to the predominance of the
pseudobase carbinol species.
At pH values above 6.0, both the pseudobase carbinol and quinoidal anhydrobase structures
can form the cis-chalcone species. The formation of this occurs with the rupture of the
heterocyclic ring which, depending on the type of anthocyanin, can make the reaction
irreversible. The formation of cis-chalcone from quinoidal anhydrobase can occur in two
different ways: directly, resulting from a sudden increase in pH, or with the formation of
ionized anhydrobase species, possibly resulting from a gradual increase in the base between
pH values 6.5 and 9. When anthocyanins ionization begins, anhydrobase structures are
formed that exhibit blue color. In an extremely alkaline environment, there is a balance
between ionized forms of cis and trans chalcones, with a yellowish color. Building on
previous work, together with recent studies, it is suggested that the possible structural
transformations of anthocyanins in aqueous medium as a function of pH can be represented
by Figure 11.

Figure 11: Possible structural transformations of anthocyanins in aqueous medium

Heating is a factor that accelerates the degradation of anthocyanins. In the presence of
cations of Al, Fe, Sn and other metals, anthocyanins form insoluble products that, in the case
of aluminum, find applications as dyes that present stability to heat, pH and oxygen superior
to that of free anthocyanins. In addition to pH, light is another factor of great importance in
changing the color of anthocyanins. The transformation is most intense when the light factor
is combined with the effect of oxygen. Anthocyanins can also be combined with HSO -
present in many foods forming colorless products from its connection with carbon 4 of
anthocyanin. The anthocyanins stability to decolorization is considerably increased by the
presence of phenolic acids. The same effect is observed by the presence of non-anthocyanin
flavonoids, especially flavonols, such as rutin. Compounds such as acetaldehyde, amino
acids, tannins, among others, also increase the stability of the molecule. This increase in
stability is attributed to copigmentation, that is, an association between anthocyanin and
flavonol by hydrogen bonds, so that flavonol will form a protective structure involving
anthocyanin.
In identifying a constituent of a plant, once it has been isolated and purified, it is necessary to
first determine the class of the compound and then find out which particular substance is
present within that class. For anthocyanins, the class of compounds can be easily
distinguished by staining tests. As an initial step for the study of anthocyanins, the detection
of the presence of pigment in the sample can be performed using the paper chromatography
technique, where the presence or absence of the anthocyanin pigment can be determined
based on areaction involving reversible change in color as a function of pH. For this, the first
step is to obtain the plant extract. Anthocyanins are unstable in solutions with neutral or
alkaline pH, and even in solutions with acidic pH the color may gradually disappear when the
solution is exposed to light. As these pigments are soluble in polar solvents, they must be
extracted using alcoholic solutions of methanol or ethanol containing acetic acid or
hydrochloric acid. The acid used in the solution decreases the pH, preventing the degradation
of unacylated anthocyanins. Most studies use alcoholic methanol solutions acidified with
HCl.

Figure 12: HPLC method

However, in food analysis the methanol solution must be replaced with ethanol, due to
methanol toxicity. After macerating an adequate amount of sample soaked in the extracting
solution, an extract sufficiently concentrated for direct application in paper chromatography
can be obtained. In case of need for storage, the extract should be kept in a dark place and,
preferably, at low temperatures.
Chromatography on paper is a liquid-liquid partition separation technique: cellulose consists
of several units of anhydrous glucose linked by oxygen atoms, so that a polar liquid such as
water has great affinity for the hydroxyls of each glucose, forming hydrogen bonds, being
retained and functioning as a stationary phase, and less polar liquids are repelled and function
as a mobile phase.
In the study of anthocyanins the most used solvent is the BAW solution. As the stationary
phase is a liquid, the separation process occurs by partition, based on the different solubilities
of the sample components in the stationary phase.
After chromatographic development, the appearance of colored spot is the first indication of
the possibility of anthocyanins in the extract. Based on the property presented by
anthocyanins to vary the color reversibly depending on the pH, it is possible to detect
whether the observed stain is due to the presence of anthocyanins or other substances, such as
betacyanins, present in beet. Betacyanins are water-soluble pigments and have a dark red
color when in a slightly acidic medium, which can cause great confusion in an initial attempt
to classify the type of pigment. The main betacyanin is betanine. However, anthocyanins and
betanines do not occur simultaneously in the same plant: some plants biosynthetically replace
anthocyanins with betanines, as is the case with plants of the family Centrospermae, like
cactus, type flowers bougainvillea, shrubs like those known as pokeweed, Phytolacca and
some ornamental plants like cat's tail. In this case, based on the change in structures resulting
from the variation of the pH of the medium, when changing the pH of the chromatogram to
slightly alkaline due to exposure to ammonia vapors, the stain should change its color. Care
must be taken not to make the medium alkaline to the point of causing the formation of
ionized chalcones which, in some cases, makes the reaction irreversible. If the substance
changes color, acid solution should be sprayed, reverting the pH to the initial value and, thus,
returning to the initial color. Betacyanins differ from anthocyanins in that they are more
unstable, so that when changing the pH to higher values betanines undergo irreversible
transformations in their structure, while anthocyanins return to their initial form.
For studies with the sole purpose of detecting the presence of anthocyanins in plant extracts,
the paper chromatography test is sufficient. In studies with the purpose of identifying
anthocyanins the chromatography on paper should be seen only as a preliminary test because,
in this case, there is a need for purification, better separation and isolation of anthocyanins.
Thin layer chromatography can be an attractive alternative to paper chromatography, since
different stationary phases can be used, allowing different separation mechanisms. The main
advantages of CCD when compared to CP are versatility and sensitivity. However, despite a
higher cost, more advanced techniques have better efficiency, better resolution and more
reliable results in separation, as is the case of high performance liquid chromatography,
capillary zone electrophoresis and chromatography high-speed counter-current.
Several methods have been used successfully for the purification of crude anthocyanin
extracts, however, the most used method today is solid phase extraction in C and Sephadex
cartridges. This is due to the relative simplicity to eliminate impurities, such as polar and
non-phenolic substances. The anthocyanin purification procedure involves applying the crude
anthocyanin extract to the cartridge containing sorbent material, followed by eluting the
individual components with appropriate solvents. Anthocyanins are strongly linked to
adsorbents by their unsubstituted hydroxyl groups. In this way, more polar substances are
eluted first than anthocyanins, such as sugars, acids and soluble substances, and later,
anthocyanin pigments are eluted.
For the separation of anthocyanins, techniques such as high performance liquid
chromatography, counter-current chromatography and capillary electrophoresis by zones
have been the most used. Paper chromatography and thin layer chromatography can also be
used in the separation step, however, they offer no other advantage than cost when compared
to, for example, the separation efficiency presented by CLAE and ECZ. When working with
large amounts of extract, an alternative is open column chromatography.
Currently, the most used technique, and one of the main ones, for the separation of
anthocyanins is HPLC with reverse phase. This type of chromatography uses sophisticated
 instruments that can be fully automated. It is a type of liquid chromatography that uses closed
columns, filled with specially prepared materials and a mobile phase that is eluted at high
pressures, presenting the ability to separate a large amount of compounds present in various
types of samples, on a time scale minutes, with high resolution, efficiency and sensitivity. In
addition to allowing excellent separation, this technique allows simultaneously to separate,
identify and quantify anthocyanin pigments without requiring excessive purification of the
extracts.
Another technique that can be employed in the separation of anthocyanins is high-speed
counter-current chromatography, described as a separation process in which a liquid phase is
retained in the column spiral by centrifugal force, while a second liquid phase immiscible
passes continuously through it. As it is a liquid-liquid support-free technique, the solute
retention is determined exclusively by its partition coefficients, and the problem of solute
adsorption in the stationary phase is eliminated. However, CLAE has better resolution and
shorter analysis time than the HSCCC technique. Anthocyanins found in nature are mobile in
an electric field. Based on this fact, capillary electrophoresis, which is a relatively new
analytical tool, it can also be used in the separation of anthocyanins.
Capillary electrophoresis has the advantages of high sensitivity, high resolution, low
sample consumption and minimal waste generation. However, when compared to HPLC, it
presents less sensitivity and less efficiency in separation of anthocyanins in complex
samples, which should be the limiting factor, resulting in the low amount of published works
using this technique for the separation of anthocyanins.
2. Gas chromatography
Gas chromatography is another technique that can be used to separate anthocyanins. In this
technique, the separation is based on the different distribution of the sample substances
between a solid or liquid stationary phase and a mobile gas phase. According to the type of
stationary phase used, gas chromatography can be classified into gas-solid chromatography,
connected phase chromatography and gas-liquid chromatography, the latter being the most
widely used form of gas chromatography. This technique is mostly used for the separation of
gases or volatile substances, however, due to the fact that anthocyanins are not volatile, there
is a need for derivatization of pigments, a process that makes the use of this technique
difficult.

Figure 13: Gas chromatography method

3. 1H NMR spectroscopy
 Proton nuclear magnetic resonance (proton NMR, hydrogen-1 NMR, or 1H NMR) is the
application of nuclear magnetic resonance in NMR spectroscopy with respect to hydrogen-1
nuclei within the molecules of a substance, in order to determine the structure of its
molecules. In samples where natural hydrogen (H) is used, practically all the hydrogen
consists of the isotope 1H (hydrogen-1; i.e. having a proton for a nucleus).
4. 13C NMR spectroscopy
Carbon-13 (C13) nuclear magnetic resonance (most commonly known as carbon-13 NMR or
13C NMR or sometimes simply referred to as carbon NMR) is the application of nuclear
magnetic resonance (NMR) spectroscopy to carbon. It is analogous to proton NMR (1H
NMR) and allows the identification of carbon atoms in an organic molecule just as proton
NMR identifies hydrogen atoms. As such 13C NMR is an important tool in chemical
structure elucidation in organic chemistry. 13C NMR detects only the 13 C isotope of carbon,
whose natural abundance is only 1.1%, because the main carbon isotope, 12C, is not
detectable by NMR since its nucleus has zero spin.
5. Diode array detection systems (DAD)
Diode-Array Detection (DAD) or Photodiode-Array Detection (PDA) is an analytical
technique that can be used to determine the purity of an analyte or related impurity peak
eluting during an HPLC separation.
The diode array detector uses the same principles of operation as a variable wavelength
detector (VWD). However, the array of diodes enables simultaneous acquisition across a
range of wavelengths, rather than just a single one. Spectral acquisition used in conjunction
with a chromatographic separation is a technique that allows the analyst to collect multiple
spectra across a chromatographic peak. Once these spectra have been collected from an
HPLC analysis, they can be used to perform an assessment of spectral peak purity
mathematically and possible identification.
6. Ultraviolet–visible spectroscopy
Ultraviolet–visible spectroscopy or ultraviolet–visible spectrophotometry (UV–Vis or
UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in part of the
ultraviolet and the full, adjacent visible spectral regions. This means it uses light in the
visible and adjacent ranges. The absorption or reflectance in the visible range directly affects
the perceived color of the chemicals involved. In this region of the electromagnetic spectrum,
atoms and molecules undergo electronic transitions. Absorption spectroscopy is
complementary to fluorescence spectroscopy, in that fluorescence deals with transitions from
the excited state to the ground state, while absorption measures transitions from the ground
state to the excited state.
7. Mass Spectrometry
Mass Spectrometry (MS) is an analytical chemistry technique that helps identify the amount
and type of chemicals present in a sample by measuring the mass-to-charge ratio and
abundance of gas-phase ions. In this instrumental technique, sample is converted to rapidly
moving positive ions by electron bombardment and charged particles are separated according
to their masses. Mass spectrum is a plot of relative abundance against the ratio of
mass/charge (m/e). These spectra are used to determine the elemental or isotopic signature of
a sample, the masses of particles and of molecules, and to elucidate the chemical structures
of molecules and other chemical compounds.
 Figure 14: Mass spectrometry method
1. In this technique, molecules are bombarded with a beam of energetic electrons.
2. The molecules are ionized and broken up into many fragments, some of which are positive
ions. Each kind of ion has a particular ratio of mass to charge, i.e. m/e ratio (value).
3. For most ions, the charge is one and thus, m/e ratio is simply the molecular mass of the
ion.
4.The ions pass through magnetic and electric fields to reach detector where they are detected
and signals are recorded to give a mass spectra.
Instrumentation and Steps of Mass Spectrometry (MS)

Figure 15: Working principle of MS

Working of Mass Spectrometry (MS)
In a typical procedure, a sample, which may be solid, liquid, or gas, is ionized, for example
by bombarding it with electrons. This may cause some of the sample’s molecules to break
into charged fragments. These ions are then separated according to their mass-to-charge ratio,
typically by accelerating them and subjecting them to an electric or magnetic field: Ions of
the same mass-to-charge ratio will undergo the same amount of deflection.
The ions are detected by a mechanism capable of detecting charged particles, such as an
electron multiplier. Results are displayed as spectra of the relative abundance of detected ions
as a function of the mass-to-charge ratio. The atoms or molecules in the sample can be
identified by correlating known masses (e.g. an entire molecule) to the identified masses or
through a characteristic fragmentation pattern.
A. Sample Inlet
Sample stored in large reservoir from which molecules reaches ionization chamber at low
pressure in steady stream by a pinhole called “Molecular leak”.
B. Ionization
Atoms are ionized by knocking one or more electrons off to give positive ions by
bombardment with a stream of electrons. Most of the positive ions formed will carry charge
 of +1. Ionization can be achieved by: Electron Ionization (EI-MS); Chemical Ionization (CI-
MS) or Desorption Technique (FAB).
C. Acceleration
Ions are accelerated so that they all have same kinetic energy. Positive ions pass through 3
slits with voltage in decreasing order. Middle slit carries intermediate and finals at zero volts.
D. Deflection
Ions are deflected by a magnetic field due to difference in their masses. The lighter the mass,
more they are deflected. It also depends upon the no. of +ve charge an ion is carrying; the
more +ve charge, more it will be deflected.
E. Detection
The beam of ions passing through the mass analyzer is detected by detector on the basis of
m/e ratio.
When an ion hit the metal box, charge is neutralized by an electron jumping from metal on to
the ion.
Types of analyzers: Magnetic sector mass analysers; Double focussing analysers; Quadrupole
mass analysers; Time of Flight analysers (TOF); Ion trap analyser; Ion cyclotron analyser.

8. Hydrolysis techniques
Can serve as a complement for the identification of anthocyanins, assisting in the separation
of anthocyanins from their sugars and bound acids. Acid hydrolysis is usually carried out
with concentrated hydrochloric acid at relatively high temperatures, causing the breakdown
of glycosidic bonds, leading to the appearance of sugars and their anthocyanidins.
Anthocyanidins and sugars can then be identified by techniques such as paper
chromatography. For the identification of anthocyanidins, comparison with standards is
desirable. In the case of alkaline hydrolysis, the rupture between the bonds of the acyl groups
is caused. The analysis of these groups can be performed after solvent extraction by
techniques such as spectroscopy and chromatography.

Figure 16: Sequential hydrolysis of typical grape anthocyanin

CHAPTER 3
COMPARISON OF THE PRESENTED ANALYTICAL PROCEDURES
Once separated and purified, anthocyanins can be identified by various techniques, the most
used of which are carbon nuclear magnetic resonance (NMR) spectrometry 13 C) and proton
(NMR) 1 H), and mass spectrometry (MS), which, in most cases, appears combined with
HPLC. The introduction of diode array detection systems (DAD) and their use when coupled
with HPLC opened new perspectives for the separation, identification and quantification of
anthocyanins, due to the possibility of obtaining complete spectra in the ultraviolet / visible
region during chromatographic analyzes. The anthocyanin detection and identification
methods, in addition to UV-Vis spectrophotometry techniques, may involve mass
spectrometry (MS) detection and nuclear magnetic resonance (NMR) detection systems.
For the identification of anthocyanins in foods and plants, two procedures can be used: direct
comparison, when standards are available, or indirect comparison, if there is no standard
material. In this case, careful comparison with data from the literature may be acceptable.
The indirect comparison, although it is an error-prone method, is justified by the lack of
standards and the long and laborious procedure for identification. In the case of a new
species, the use of techniques such as HPLC, NMR 13 C and NMR 1H, MS, in addition to
the combination of these techniques, can provide information that helps in the
characterization of the pigment. The HPLC technique is in most cases only in the separation
stage, as there is a great difficulty in the identification of anthocyanins, not only in HPLC,
but in all other techniques, due to the lack of anthocyanin pigment patterns.
In order to minimize the difficulty of the lack of standards and laborious procedures for the
identification of anthocyanins, in 1999, Goiffon proposed a method for identification based
on parameters that affect the retention of liquid chromatography. In the proposed
methodology, the authors relate the retention time of several anthocyanins with the retention
time of an anthocyanin that is present in most red fruits: cyanidin-3-glucoside. It is suggested
that this relationship between retention times (α) it would be the way to identify the different
anthocyanins. Thus, the identification of anthocyanins in fruits is considerably facilitated,
minimizing the problem of lack of standards. They determined the value of α for 40
anthocyanins and anthocyanidins using 2 types of eluents, while in most of the works
described in the literature for anthocyanins identification, only one type of mobile phase is
cited.
UV-Vis spectrophotometry is a very important auxiliary tool in anthocyanin analysis,
especially when coupled with techniques such as HPLC. Anthocyanins have different
spectral profiles depending on pH, due to the predominance of different structures in each
medium. According to some researchers, such as Francis, when anthocyanidin and sugar(s)
are (are) known, spectral data can be very useful tools in determining the position of the
glycosidic bond. This author states that when an anthocyanin has a sugar molecule attached
to the carbon of position 5, in an acidic environment, a shoulder is observed in the absorption
band at 440 nm, in addition to the flavylic cation band at 520 nm. However, even serving as a
complementary tool for qualitative analysis, when used in conjunction with HPLC analysis,
UV-Vis spectrophotometry, when applied alone, is more useful in quantitative analysis.
An interesting application of UV-Vis spectrophotometry in the field of anthocyanin
identification was carried out in 2000 by Cabrita, where it was proposed to identify groups of
anthocyanins. The authors report that anthocyanins can be divided into two large groups
according to the curve profile obtained at the maximum absorption wavelength of each
anthocyanin as a function of pH. The characteristic curves of each group can be explained by
the substituting groups in ring B of the fundamental structure of anthocyanins. The number
of oxygenated substituents (hydroxy or methoxy) in ring B causes a bathochromic effect at
relatively low pH values, as well as at high pH values. Thus, the hydroxy and methoxy
groups have similar effects on the bluish chromophoric forms present in the balance. In this
work strawberries, rice, Korean Abies and blueberries as sources of anthocyanins, and they
underwent purification and isolation processes with reverse-phase HPLC for subsequent
identification using nuclear magnetic resonance, proving that spectrophotometry is an
excellent complementary tool in anthocyanin analysis. Even presenting a relatively low cost
advantage when compared to the most advanced techniques, due to the large amount of
substances present in a plant extract, it cannot be reliably stated about the structure of a
pigment just by observing the obtained UV-Vis spectrum.
One technique that can provide excellent information on the molecular formula of an
anthocyanin is mass spectrometry (MS). Mass spectrometry, in essence, consists of the
generation of ions by the fragmentation of the sample molecules and the detection of
fragments is done according to the massa The positively charged ions produced are
accelerated in a magnetic field that disperses and allows relative measurements of ions of
different mass / charge ratios. The result of measuring ion abundance versus mass constitutes
the mass spectrum, which consists of a series of lines varying in intensity in different units of
mass. For the analysis of anthocyanins, sources of rapid bombardment of atoms are generally
used (Fast Atom Bombardment, FAB) and electron spray (Electrospray Ionization, ESI). An
important characteristic of ESI is the fact that the samples must be introduced as a solution,
making possible the coupling with many separation techniques, such as HPLC. The
advantage of the mass spectrometry technique is the ability to indicate the exact molecular
formula of the substance without the need for large amounts of sample, as the technique can
work with trace amounts. However, care should be taken with the presence of isomers, which
is probably its limiting factor. The best way to prevent this error should be in the purification
and isolation stages, after detecting the presence of anthocyanins in the extract.
With the possibility of using advanced equipment that provide information such as mass
spectrum and carbon NMR and protons, the procedure for elucidating the structure of an
anthocyanin should, in its first stage, lead to the recognition of the molecular formula, as you
can get a general idea of the molecule (that is, the number and type of atoms). Obtaining the
molecular formula begins with the location of the peak of the molecular ion obtained by the
mass spectrum. From this point the NMR spectra can bring information about the location of
the carbon and hydrogen atoms in the molecule's structure. It is observed that the reliability
of the results will be related to the purity achieved.
Nuclear magnetic resonance (NMR) spectroscopy can provide information for determining
the structure of an organic compound, by measuring the magnetic moments of carbon and
hydrogen atoms. NMR spectroscopy is basically another form of absorption spectroscopy,
where, under appropriate conditions in a magnetic field, a sample can absorb electromagnetic
radiation in the radiofrequency region at a frequency governed by the structural
characteristics of the molecule, the absorption being a function of certain nuclei of the
molecule. The most efficient NMR spectroscopy technique for structural elucidation is two-
dimensional NMR. The two dimensions referred to in the 2-D spectrum are the frequency
axes, where the experiment requires two Fourier transforms perpendicular to each other in
two independent time axes, which lead to two orthogonal frequency axes. One of the biggest
advantages of working with NMR compared to techniques such as, for example, HPLC in the
identification of anthocyanins is that there is no need for standards. However, the difficulties
for this method are in the complexity of the spectra and the influence of the solvent.
The coupling of different instrumental techniques constituting hyphenated instruments or
second-order techniques, such as liquid chromatography with detection by mass spectrometry
or detection in the UV-Vis region through a diode arrangement system, has been increasingly
applied in analytical chemistry laboratories because it allows obtaining additional
information from the correlation of different responses, such as, for example, an absorption
spectrum and a chromatographic profile, obtained simultaneously from the same sample. The
use of hyphenated techniques, combining separation and identification of anthocyanins, is
common. Thus, the identification of anthocyanins is facilitated by the amount of information
that can be made available by this equipment. This is due to the high selectivity,
improvement in the determination limit and reduction in the analysis time.
The rapid and continuous development in instrumentation in recent years, particularly those
involving nuclear magnetic resonance spectroscopy and mass spectrometry, has provided
considerable impetus for structural elucidation in all fields of natural product chemistry. For
anthocyanins, this means that extremely complex structures can be determined. In general,
these determinations do not require continuous derivatization or degradation, however, the
use and maintenance of more sophisticated equipment significantly increases the cost, thus
limiting its users and applications.
One of the greatest difficulties in spectrophotometric resolution of chemical systems is the
estimation of the number, concentration and spectra of the species involved in the system.
Different multivariate statistical techniques have been used to find the best methodology to
extract information from these systems, in order to identify the species present and to
qualitatively and quantitatively determine the concentrations of some or all of them. These
techniques are known as chemometric methods. The applicability of each methodology
depends on the data set (experimental information) submitted to the analysis. In recent
research, chemometric methods associated with spectrophotometric data have been used to
assist in the identification of anthocyanins extracted from flowers of the genus Hibiscus. In
these works, it is observed that with the application of chemometric methods in
spectrophotometric data, it was possible to identify anthocyanins without the use of very
expensive techniques: balances between different forms of anthocyanins were identified,
where the values calculated for the equilibrium constants are found in excellent agreement
with values suggested by the literature.
CHAPTER 4
CONCLUSIONS
In general, it is observed that one of the greatest difficulties in identifying individual
anthocyanins is the lack of standards, and that the choice of the method to be used depends
on the purpose of the analysis. To detect the presence of anthocyanins in a plant, a simple test
using paper chromatography may be sufficient. Optical methods like UV-Vis are very useful
complementary tools for the characterization of anthocyanins. Identifications of individual
anthocyanins require more advanced methods such as HPLC, NMR spectroscopy and mass
spectrometry. The combination of these techniques can bring enough information for the
complete elucidation of the structure of an anthocyanin, however, care must be taken with
isotope analysis in the case of MS, when choosing the solvent in NMR. According to
research carried out to date, it is clear that there is no defined criterion for the
characterization of new anthocyanins present in new plants.
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