THESIS. Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Transcription

1 Commercial Applicability of an Innovative Anthocyanin Purification Technique, Utilizing Mixed-Mode Solid-Phase Extraction THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Steven Tyler Simmons Graduate Program in Food Science and Nutrition The Ohio State University 2012 Master's Examination Committee: Professor M. Monica Giusti, Advisor Professor W. James Harper Professor Jeff Culbertson

2 Copyrighted by Steven Tyler Simmons 2012

3 ABSTRACT Anthocyanins represent a class of natural pigments that produce an extensive array of colors in plant tissues, and have been associated with the ability to elicit health beneficial properties related to their antioxidant ability. There has been increased interest in expanding the use of anthocyanins as natural food colorants, in response to legislative action and negative consumer perception in association with synthetic dyes. Researchers in our lab recently developed a patent-pending, rapid, economical, and high throughput solid-phase anthocyanin extraction (SPE) method, utilizing the Oasis MCX mixed-mode cation-exchange SPE cartridge. The new method outperformed currently used SPE techniques for anthocyanin isolation at bench top scale (single run), and may prove applicable for the commercial production of large volumes of highly pure anthocyanin mixtures to be used as natural colorants or for research purposes. The objective of this study was to explore the potential for commercial application of the patentpending mixed-mode anthocyanin purification method. In order to achieve this goal the robustness of the method was assessed with previously examined and new pigment sources, its compatibility with other commercially available mixed-mode SPE resins was investigated, sorbent capacity was quantified, and cartridge longevity was analyzed. Anthocyanin isolate purity was also evaluated via molar absorptivity calculations, utilizing Lambert-Beer s law. Various quantities of several anthocyanin containing crude extracts were purified by the proposed technique in order to validate the method, and assess sorbent holding capacity. Sequential purification procedures (up to 20) were conducted on fresh and used cartridges with newly prepared, as well as frozen crude extracts, to assess cartridge longevity and commercial applicability. Purified anthocyanin extracts as well as phenolic constituents were analyzed via high-performance liquid chromatography (HPLC) coupled to a photodiode array (PDA) detector. ii

4 UV-Visible spectrophotometry was conducted for the determination of total phenolics, total monomeric anthocyanins, and anthocyanin isolate purity, utilizing Folin-Ciocalteau (FC) colorimetry, the ph differential method, and molar absorptivity calculations, respectively. The mixed-mode anthocyanin purification method showed robustness under the conditions used, producing extracts of extremely high purity (some > 99%, based on HPLC-PDA percent area under the curve) regardless of the cartridge utilized (P>0.05). Neither cartridge significantly (P>0.05) outperformed the other in terms of capacity (0.54mg anthocyanins/500mg sorbent) or longevity, indicating the compatibility of the method with different cationexchange/reversed-phase sorbents, and showing promise for commercial application. Significantly lower (P<0.01) isolate purities were calculated by spectrophotometric analysis utilizing molar absorptivities, indicating the presence of other materials that do not absorb in the UV-visible range and cannot be detected by the PDA. The proposed method allowed for the simple isolation of high-purity anthocyanin extracts. The increased availability of high-purity anthocyanin mixtures may promote their incorporation in food. The mixed-mode sorbents, regardless of manufacturer, showed the ability to contribute to numerous purifications while maintaining excellent isolation ability, further strengthening the potential for application on a commercial scale. A scale-up of the cartridge size may benefit the food colorant and nutraceutical industries, as well as the cartridge manufacturers, by promoting the production of high-purity anthocyanin mixtures. iii

5 ACKNOWLEDGEMENTS I would first like thank my advisor Dr. M. Monica Giusti for her constant encouragement and expertise in the field of Food Science. Fulfillment of this research would have been impossible without her willingness to share her vast knowledge on anthocyanins. Many thanks to Dr. W. James Harper and the entire Food Science & Technology faculty for providing me with the foundation of knowledge necessary for me to achieve my academic goals. The learning opportunities available through their coursework are second to none, and I am extremely fortunate to have been taught by the very best. I would also like to thank my colleagues and lab mates for always being there for me. Graduate school and life in general can be stressful at times and we all need one another to be successful. I wish to thank my family and friends for their constant love and encouragement. The joy that they brought me each and every day pushed me to accomplish feats that I had not thought possible. Lastly, a special thanks to Dr. Jeff Culbertson for his constant guidance and support throughout my collegiate career. He saw something in me that I did not know existed, and I will never be able to thank him enough for providing me with the opportunity to pursue a graduate degree in Food Science. iv

6 VITA February 27, Born Columbus, Ohio USA B.S. in Agriculture majoring in Food Business Management with a minor in Entrepreneurship, The Ohio State University, Columbus, OH 2010 to Graduate Research Associate, Department of Food Science & Technology, The Ohio State University, Columbus, OH FIELD OF STUDY Major Field: Food Science and Nutrition v

7 TABLE OF CONTENTS Page ABSTRACT... ii ACKNOWLEDGEMENTS... iv VITA... v FIELD OF STUDY... v TABLE OF CONTENTS... vi LIST OF TABLES... ix LIST OF FIGURES... x CHAPTER 1: LITERATURE REVIEW COLOR What is color Color in food ANTHOCYANINS Function of anthocyanins Structure of anthocyanins Glycosylation and acylation The effect of ph on anthocyanin structure Stability of anthocyanins vi

8 1.3 SOLID-PHASE EXTRACTION (SPE) Principles of SPE Reversed-phase technique Mixed-mode technique PRESENT STUDY CHAPTER 2: MATERIALS AND METHODS REAGENTS ANTHOCYANIN SOURCES AND EXTRACT PREPARATION Plant materials Sample preparation Chemical extraction SPE CARTRIDGES AND PURIFICATION METHOD MCX and X-C SPE C 18 SPE Method validation Cartridge capacity Cartridge longevity SAMPLE ANALYSIS HPLC-PDA UV-Visible spectrophotometry STATISTICAL ANALYSIS vii

9 CHAPTER 3: RESULTS AND DISCUSSION METHOD VALIDATION Pigment isolation and determination of putity CARTRIDGE CAPACITY Total phenolics Total monomeric anthocyanins Sorbent holding capacity CARTRIDGE LONGEVITY Resin endurance Resin effectiveness following storage Commercial applicability Calculation of purity utilizing molar absorptivities CHAPTER 4: CONCLUSIONS REFERENCES APPENDIX viii

10 LIST OF TABLES Table Page 3.01: Total phenolic and total monomeric anthocyanins concentrations of three crude extracts in terms of GAE : Quantity of phenolics and anthocyanins of three crude extracts loaded onto mixed-mode sorbents : Mixed-mode sorbent anthocyanin holding capacity in relation to total phenolics and monomeric anthocyanins of three crude extracts : Comparison of calculated purity of blackberry anthocyanins isolate by % AUC and molar absorptivity calculations A.01: Achieved anthocyanin fraction purity using the proposed method, anthocyanin content, and approximate market price of various anthocyanin containing commodities ix

11 LIST OF FIGURES Figure Page 1.01: The electromagnetic spectrum : Perception of color: light source, object, and observer : CIELAB color space : General structures of a chlorophyll, anthocyanin, carotenoid, and betalain : Flavylium cation : The six most common anthocyanidins, arranged in deepening of hue : Total numbers of anthocyanins containing various monosaccharides : Example of an acylated anthocyanin : Total numbers of anthocyanins containing various acids : The effect of ph on anthocyanin structure : Intermolecular and Intramolecular stacking of acylated anthocyanins : Oasis MCX sorbent : Strata X-C sorbent : HPLC-PDA chromatograms of purified blueberry, red cabbage, red table grape, and strawberry extracts observed at nm max-plots : HPLC-PDA chromatograms of purified blueberry extract, anthocyanins observed at nm and overall composition observed at nm : Comparison of the calculated purity of four commodities between studies : HPLC-PDA max-plot chromatograms of crude and purified blue potato extract : HPLC-PDA chromatograms of purified blackberry, eggplant, and blue potato extracts observed at nm max-plots x

12 3.06: Isolate purity (based on % AUC of HPLC-PDA chromatograms) achieved for blackberry, eggplant, and blue potato crude extracts : FC colorimetry: GAE vs. absorbance at 765nm : Calculation of anthocyanin loss in phenolic fraction : Blackberry pigment loss vs. pigment loaded : Red table grape pigment loss vs. pigment loaded : Strawberry pigment loss vs. pigment loaded : Blackberry anthocyanins isolate purity. Successive purifications with fresh extract and fresh mixed-mode sorbents : Blackberry anthocyanins isolate purity. Successive purifications with extract stored frozen 2 weeks and fresh mixed-mode sorbents : Blackberry anthocyanins isolate purity. Successive purifications with extract stored frozen 2 weeks and used mixed-mode sorbents stored in MeOH 2 weeks : Max-plots of blackberry anthocyanin fractions from runs 1, 5, 10, 15, and 20 as compared to the ideal chromatogram produced by a pure blackberry anthocyanins mixture. Purification through the Oasis MCX SPE cartridge : Max-plots of blackberry anthocyanin fractions from runs 1, 5, 10, 15, and 20 as compared to the ideal chromatogram produced by a pure blackberry anthocyanins mixture. Purification through the Strata X-C SPE cartridge : Blackberry anthocyanins isolate purities across 20 purifications xi

13 CHAPTER 1: LITERATURE REVIEW 1.1 COLOR WHAT IS COLOR Color is a sensation experienced by an individual when the retina of the eye receives radiant energy between the wavelengths of 380 and 770nm, otherwise referred to as the visible spectrum (Wrolstad and Smith 2010). The visible spectrum encompasses only a small portion of the electromagnetic spectrum, and is flanked by ultraviolet light at lower wavelengths and infrared light at higher wavelengths (Konica Minolta 2007). The visible spectrum includes seven major colors: violet, indigo, blue, green, yellow, orange, and red. Figure 1.01 provides an illustration of the electromagnetic spectrum, with emphasis on the visible wavelengths of light. 1

14 Figure 1.01 The electromagnetic spectrum. (Konica Minolta 2007) In order for color to be perceived there must be a light source within the visible spectrum, a pigmented object, and an observer (Konica Minolta 2007). Figure 1.02 demonstrates this three element relationship, where the light source is sunlight, the object is an apple, and the observer is 2

15 a human eye. Exclusion of any one of these three elements would result in the inability to perceive color. Figure 1.02 Perception of color: light source, object, and observer. A specific color is created through the combination of three attributes: hue, lightness, and saturation (Konica Minolta 2007). Hues form the color wheel and are commonly referred to as primary colors. The color wheel includes red, red-purple, purple, blue-purple, blue, blue-green, green, yellow-green, yellow, and orange. Lightness is the difference between brightness and darkness or the amount of white or black present in a color. Saturation is the difference between vividness and dullness. Color, in addition to texture and flavor, is a principal quality attribute for all food products (Wrolstad and Smith 2010). The appearance of a food product is the first and most influential attribute for prospective customers (Giusti and Wrolstad 2003). Humans have the ability to detect millions of different colors, but cannot accurately recall previously observed colors due to poor color memory (Wrolstad and Smith 2010). Additionally, color is a sensation 3

16 that is perceived differently by each individual, and in order to standardize all perceivable colors a variety of color spaces have been developed. CIELAB color space is one of the most commonly used color spaces for measuring the color of food products (Konica Minolta 2007; Hunter Associates Laboratory 2008). Figure 1.03 depicts the spherical diagram of CIELAB color space, where hue is represented around the circumference (+a = redness, -a = greenness, +b = yellowness, -b = blueness), lightness is represented by either pole (+L = whiteness, -L = blackness), and saturation is represented by the distance from the center (increasing ±a or ±b). Figure 1.03 CIELAB color space. (Konica Minolta 2007) 4

17 In order to assure the color quality of food products food companies often use equipment such as colorimeters. Colorimeters allow for simple, rapid, and accurate quantification of color using a wide variety of color spaces (Konica Minolta 2007). Colorimeters are used to quantify the color of a variety of food products, from naturally pigmented bananas and apples, to artificially colored products such as candies and beverages COLOR IN FOOD There are four categories of food colors which include natural colors, nature-identical colors, synthetic colors, and inorganic colors (Mortensen 2006). Natural colors or pigments are color imparting substances that occur naturally in plant and animal cells and tissues (Giusti, Schwartz et al. 2008; Mortensen 2006). Gold, titanium dioxide, and silver are examples of inorganic colors (Mortensen 2006). Nature-identical colors are man-made pigments which are structurally identical to those found in nature. Synthetic colors are also man made, but do not also occur naturally, and have been scrutinized for their potentially negative side effects (Andersen and Jordheim 2006). Numerous synthetic food dyes have been linked with hyperactivity, attention-deficit/hyperactivity disorder (ADHD), and behavioral problems in children (Jacobson 2008). The European Food Safety Authority is currently evaluating research that shows that some food colorants potentially cause increased hyperactivity in children, and their findings may influence the United States Food and Drug Administration to re-evaluate the safety of certified synthetic colorants (Sherma, Markow et al. 2011). Myoglobin is the primary pigment responsible for the color of meat. The secondary pigment in meat is hemoglobin, the pigment found in blood. Myoglobin and hemoglobin are heme compounds, the most abundant colored compounds found in animal tissues. The overwhelming majority of naturally occurring pigments are found in plant cells and tissues (Giusti, Schwartz et al. 2008). Many of these compounds have functions other than acting as 5

18 colorants, possibly the most important of which is the ability to exhibit antioxidant activity (Giusti and Wrolstad 2003). The four main classes of natural pigments found in plants (i.e. fruits and vegetables) are chlorophylls, carotenoids, betalains, and anthocyanins (Giusti, Schwartz et al. 2008). Figure 1.04 provides the general structures of each of the major plant pigments. Chlorophylls are light-harvesting pigments that are found in green plants, algae, and even a specialized subset of bacteria known as photosynthetic bacteria. Chlorophyll is a cyclic tetrapyrrole with centralized magnesium (Mortensen 2006). Carotenoids are the most widespread of the plant pigments and are responsible for colors ranging from the orange of carrots to the yellow-orange-red of leaves during autumn (Giusti, Schwartz et al. 2008). Carotenoids are present in all chlorophyll-containing tissues, and act as secondary light energy harvesting compounds and photoprotectants. The primary function of carotenoids in the diet of humans is as a precursor of vitamin A. In addition to plants carotenoids are also found in some animal tissues, such as the flesh of salmon (Mortensen 2006). Betalains represent a group of red and yellow pigments located in the vacuoles of plant cells (Giusti, Schwartz et al. 2008). The most common betalain containing vegetables are red beet and amaranth. Anthocyanins are water-soluble phenolic compounds responsible for a wide range of colors in plants (Andersen and Jordheim 2006; Giusti, Schwartz et al. 2008). Anthocyanin pigments can produce blue, purple, violet, magenta, red, and orange coloration. The word anthocyanin is derived from the Greek words anthos and kyanos meaning flower and dark blue respectively. 6

19 (a) (b) (c) (d) Figure 1.04 General structures of (a) chlorophyll, (b) anthocyanin, (c) carotenoid, & (d) betalain. 1.2 ANTHOCYANINS FUNCTION OF ANTHOCYANINS According to Kong, Brouillard, et al. (2003) the main function of anthocyanins, in nature, is to provide color to plants and plant materials in order to attract animals and insects for pollination and seed dispersal. Additionally, anthocyanins act as photoprotectants, antioxidants, antimicrobial, and antibacterial agents in plant tissues (Kong, Brouillard et al. 2003; Gould and Lister 2006). Throughout the world there has been interest in anthocyanins as natural food colorants, in response to legislative action and consumer preferences in association with synthetic dyes 7

20 (Andersen and Jordheim 2006). Furthermore, anthocyanin consumption has been associated with a probable reduction in the risks of chronic diseases such as cancers, cardiovascular diseases, viral infections, and even Alzheimer s (Andersen and Jordheim 2006; He and Giusti 2011). The ability of anthocyanins, along with other fruit and vegetable polyphenolics, to elicit protective properties such as anti-allergic, anti-inflammatory, anti-proliferative, and anti-mutagenic effects, as well as microcirculation improvement, peripheral capillary fragility prevention, diabetes prevention, and improvement of vision have been investigated over the past two decades (Ghosh and Konishi 2007). Anthocyanins are popularly regarded as nutraceuticals mainly due to their antioxidant ability, and have been used for years in nutritional supplements (Andersen and Jordheim 2006; He and Giusti 2011). Most bioassays on anthocyanins have been performed using crude anthocyanin extracts due to the fact that isolation of pure anthocyanins is time consuming and expensive (He and Giusti 2011). These crude extracts contain impurities, such as other phenolics, which are biologically active and may become confounding factors in anthocyanin centered biological studies. As a result, the determination of the health benefits of anthocyanins may be vague (He and Giusti 2011; Seeram, Adams et al. 2004) STRUCTURE OF ANTHOCYANINS Anthocyanins are members of the flavonoid subgroup of phenolic compounds due to their C 6 C 3 C 6 carbon skeleton (Rodriguez-Saona and Wrolstad 2001). Figure 1.05 depicts the most elementary structure of anthocyanins, the flavylium cation. The anthocyanin molecule consists of an aglycone base, or anthocyanidin, often one or more sugar, and frequently one or more acyl acid (Andersen and Jordheim 2006; Francis and Markakis 1989). Glucose, rhamnose, xylose, galactose, arabinose, fructose, glucuronic acid, and di- and trisaccharides of these sugars are the most common anthocyanin substituents (Francis and Markakis 1989; Giusti, Schwartz et al. 8

21 2008). If an acyl group is present it will typically attach to one of the sugar residues on the anthocyanin, and is most commonly an aromatic acid including p-coumaric, caffeic, ferulic, sinapic, gallic, or p-hydroxybenzoic acid, or an aliphatic acid such as malonic, acetic, malic, succinic, or oxalic acid (Giusti, Schwartz et al. 2008). + Figure 1.05 Flavylium cation. R 1 and R 2 = H, OH, or OCH 3, R 3 = glycosyl, R 4 = H or glycosyl. Ninety percent of all anthocyanins are comprised from six aglycones or anthocyanidins including pelargonidin (Pg), cyanidin (Cy), delphinidin (Dp), peonidin (Pn), petunidin (Pt), and malvidin (Mv), all of which occur commonly in foods (Andersen and Jordheim 2006; Kong, Brouillard et al. 2003). The type of anthocyanin and the color produced by that pigment is resultant of the number of hydroxyl or methoxy groups, the type, number, and site of attached sugars, and the type and number of acyl acids attached to the sugars (Giusti, Schwartz et al. 2008; Giusti and Wrolstad 2003). Figure 1.06 provides the structures of the six most common anthocyanidins as well as their general color under acidic conditions. The figure illustrates the ability of methoxy and hydroxyl groups to deepen hue (redness and blueness respectively). This is achieved through increasing the compounds relative electron mobility. 9

22 Figure 1.06 The six most common anthocyanidins, arranged in deepening of hue. (Giusti, Schwartz et al. 2008) GLYCOSYLATION AND ACYLATION Glycosylation has been identified in approximately 90% of all anthocyanins (Andersen and Jordheim 2006). Monosaccharide glycosyl moieties are the most common compounds attached to anthocyanins, although anthocyanins have been discovered with more complex attached sugars, and as previously stated consist of glucose, galactose, rhamnose, arabinose, xylose, fructose, and glucuronic acid. Figure 1.07 provides a general depiction of the number of anthocyanins in nature containing various monosaccharides. A large number of glycosylated anthocyanins have been discovered recently due to the increased interest in the study of natural pigments over the past two decades. Those discovered post 1992 are represented by the darkened region of each bar. The majority of glycosylated anthocyanins have attached sugars at the 3 10

23 position followed by the 5 position, and in much more rare cases the 7, 3, 5, and 4 position (Figure 1.05), in order of decreasing occurrence (Andersen and Jordheim 2006; Bakowska- Barczak 2005). Figure 1.07 Total numbers of anthocyanins containing various monosaccharides. The darkened upper portion of each bar represents the anthocyanins reported post (Adapted from Andersen and Jordheim 2006) As previously stated, the glycosyl moieties may be acylated with organic acids including a wide range of aromatic and aliphatic acylating agents (Bąkowska-Barczak 2005). More than 65% of properly identified anthocyanin structures are acylated, with the majority being acylated at the 6-OH group of a C3 sugar (Figure 1.08) (Andersen and Jordheim 2006; Giusti and Wrolstad 2003). Figure 1.09 provides a general depiction of the number of anthocyanins in nature containing various acid residues. A large number of acylated anthocyanins have been discovered recently due to the increased interest in the study of natural pigments. Those discovered within the past 20 years are represented by the darkened region of each bar. The vast majority of acylated anthocyanins are derived from vegetable tissues. 11

24 Figure 1.08 Example of an acylated anthocyanin, delphinidin-3-malonylglucoside-5-glucoside. Figure 1.09 Total numbers of anthocyanins containing various acids. The darkened upper portion of each bar represents the anthocyanins reported post (Adapted from Andersen and Jordheim 2006) 12

25 The abundance of sugars and acids attached to the structure of an anthocyanin has a great impact on the color produced by the pigment (Andersen and Jordheim 2006). Increasing glycosyl and acyl groups also improves the anthocyanins ability to solubilize due to the increased capacity to participate in hydrogen bonding. Additionally, complex glycosylation and acylation of anthocyanin molecules has shown an improvement in the stability of the pigment to a variety of conditions (Giusti and Wrolstad 2003). The stabilization is often attributed to stacking of acyl groups with the flavylium cation, protecting the anthocyanin structure from the nucleophile attack of water and the formation of the colorless carbinol pseudobase or chalcone structures (Bąkowska-Barczak 2005). This stacking is also referred to as intramolecular copigmentation (Rodriguez-Saona, Giusti et al. 1999) THE EFFECT OF ph ON ANTHOCYANIN STRUCTURE Anthocyanins have the unique property to exist in four structural forms depending on the ph of their environment (He and Giusti 2011). These forms include the positively charged flavylium cation, the hemiacetal carbinol pseudobase, the chalcone, and the negatively charged quinonoidal base (Giusti, Schwartz et al. 2008). The anthocyanins most stable form, the red flavylium cation, dominates at low ph, the colorless hemiacetal and chalcone are present at more neutral ph, and the blue quinonoidal base dominates under neutral to alkaline conditions. These transformations are structure dependent and vary depending on the specific anthocyanins present. Figure 1.10 illustrates the transformation of an anthocyanin molecule in response to changing ph. These structural changes result from kinetic and thermodynamic competition between proton transfer reactions and hydration reactions (Fossen, Andersen et al. 1998). The anthocyanin molecule becomes more unstable as ph increases as a result of these structural changes, and often loses color due to hydration at the 2 position. 13

26 Figure 1.10 The effect of ph on anthocyanin structure. (Adapted from He and Giusti 2011) The ability of anthocyanins to transform structurally depending on the ph of their environment is extremely rare for most secondary plant metabolites (He and Giusti 2011). At low ph anthocyanins are predominantly in their ionized form and are likely to form complexes with other compounds such as proteins, polysaccharides, and other flavonoids (Giusti, Schwartz et al. 2008). As ph increases and the anthocyanin s structure transforms to gain a negative electric charge, reciprocal ionic interactions are more likely to occur. The ability of anthocyanins to easily reverse conformation shows extreme promise for the evolution of anthocyanin purification. 14

27 1.2.5 STABILITY OF ANTHOCYANINS Anthocyanins are commonly considered to be relatively unstable compounds. As previously stated, anthocyanin pigments are most stable under acidic conditions and when acylated (Giusti, Schwartz et al. 2008; Turker, Aksay et al. 2004). A number of factors, in addition to the anthocyanin s structural characteristics and environmental ph, affect the color and stability of the pigments. Pigment concentration, temperature, light, oxygen, copigmentation, self-association, metal ions, enzymes, ascorbic acid, sugars, proteins, phenolic compounds, and sulfur dioxide are all factors which affect the color and stability of anthocyanins (Rodriguez- Saona, Giusti et al. 1999; He and Giusti 2011). Crude anthocyanin extracts often contain a variety of compounds, such as sugars, proteins, and phenolics, which accelerate the degradation of the pigment. Consequently, high purity anthocyanin extracts are desirable to improve pigment stability (He and Giusti 2011). Anthocyanins may also be stabilized by reducing their exposure to heat and oxygen. Heating shifts the equilibrium structure towards the colorless forms resulting in the loss of the desired red pigmentation (Giusti, Schwartz et al. 2008). Highly hydroxylated anthocyanins have the lowest thermal stability, whereas highly acylated anthocyanins are the most stable to heating (Giusti, Schwartz et al. 2008; Turker, Aksay et al. 2004). Additionally, Havlíková and Míková (1985) determined that short-time-high-temperature treatments result in less pigment degradation than long-time-low-temperature treatments for the incorporation of anthocyanins in pasteurized products. It has also been determined that processing of anthocyanin-containing products, such as juices, should be done under nitrogen or vacuum in order to minimize oxygen exposure (Giusti, Schwartz et al. 2008). The unsaturated structure of anthocyanins increases their susceptibility to interaction by molecular oxygen. Moreover, ascorbic acid and anthocyanins are mutually destructive in the presence of oxygen, making their incorporation into juices difficult 15

28 (Talcott, Brenes et al. 2003). The destructive effect of oxygen may be minimized through copigmentation in cases where oxygen removal is challenging. In nature, anthocyanins are often stabilized by copigmentation with other anthocyanins or other flavonoids, self-association, metal cations, and acylations, some of which may translate to increased anthocyanin stability as a food colorant (Andersen and Jordheim 2006; Giusti and Wrolstad 2003). Acylation of the anthocyanin molecule promotes intramolecular and intermolecular copigmentation, as well as self-association reactions, greatly improving stability (Giusti and Wrolstad 2003). Increased stability is achieved, in part, by the spatial configuration or stacking of acylated pigments (Figure 1.11). Figure 1.11 Intermolecular and Intramolecular stacking of acylated anthocyanins. (Adapted from Yoshida, Kondo et al. 1992) 16

29 1.3 SOLID-PHASE EXTRACTION PRINCIPLES OF SOLID-PHASE EXTRACTION A variety of methods have been developed in order to separate and purify anthocyanins. Various forms of chromatography including paper chromatography, thin-layer chromatography, column chromatography, countercurrent chromatography, and high-performance liquid chromatography have been used for anthocyanin isolation, as well as capillary electrophoresis and solid-phase extraction (SPE) (Takeoka and Dao 2008). SPE methods are currently most popular for anthocyanin fractionation due to their efficiency and relatively low cost, comparatively (He and Giusti 2011; Escribano-Bailón and Santos-Buelga 2003). SPE is one of the most important and frequently used sample preparation techniques for purifying complex mixtures due to its speed and cost-effectiveness (Pavlovic, Babic et al. 2010; Majors 2008). In SPE, compounds are partitioned between a solid (stationary) phase and a liquid (mobile) phase, similar to the principles of liquid chromatography. The desired analytes typically have a higher affinity for the solid phase than the other components in the sample matrix, allowing for their retention to the solid phase and the removal of other compounds with the liquid phase (Majors 2008; Escribano-Bailón and Santos-Buelga 2003). Sorbent packed cartridges are common SPE mediums. SPE has been widely used in order to remove unwanted materials such as sugars, acids, and proteins from anthocyanin extracts, but separation of non-anthocyanin phenolics has always been an issue (Kraemer-Schafhalter, Fuchs et al. 1998; He and Giusti 2011; Escribano-Bailón and Santos-Buelga 2003). Most SPE sorbents adsorb analytes non-selectively, which allows for contamination of the anthocyanin fraction with undesirable components such as phenolics (He and Giusti 2011). Until recent years sorbents consisting of C 18 chains bonded on silica have been generally considered to be most effective for anthocyanin purification (Kraemer-Schafhalter, 17

30 Fuchs et al. 1998; Rodriguez-Saona and Wrolstad 2001). Rodriguez-Saona and Wrolstad (2001) described the basic protocol for anthocyanin pigment isolation using the C 18 sorbent, indicating the production of relatively pure anthocyanin extracts. Purification by this technique often results in incomplete pigment isolation, requiring further purification steps, such as HPLC, to obtain pure anthocyanins (Escribano-Bailón and Santos-Buelga 2003) REVERSED-PHASE TECHNIQUE Anthocyanin purification using C 18 SPE utilizes a hydrophobic mechanism known as reversed-phase. The reversed-phase separation mechanism involves the combination of a nonpolar stationary phase and a polar mobile phase (Rodriguez-Saona and Wrolstad 2001; Escribano- Bailón and Santos-Buelga 2003). This allows for non-polar constituents such as anthocyanins and other phenolics to adhere to the non-polar C 18 sorbent, while polar compounds such as sugars and acids are washed away with the polar mobile phase (Rodriguez-Saona and Wrolstad 2001; Kraemer-Schafhalter, Fuchs et al. 1998). Ethyl acetate can be applied to the cartridge, following washing of the polar compounds, in order to attempt to remove the majority of the nonanthocyanin phenolics (Rodriguez-Saona and Wrolstad 2001; Jackman and Smith 1996). The remaining compounds, the majority of which are anthocyanins, are then eluted by way of acidified methanol. For the best resin affinity anthocyanins should be kept at an acidic ph in order to insure that they are in their more stable protonated form MIXED-MODE TECHNIQUE In addition to the hydrophobic mechanism of the reversed-phase technique, a second mechanism may be added to improve the selectivity of an SPE sorbent. This is referred to as mixed-mode, and typically results from the combination of ion exchange and reversed-phase 18

31 interactions (Majors 2008). Mixed-mode sorbents have been used primarily for the determination of pharmaceuticals and illicit drugs in water and urine samples (Pavlovic, Babic et al. 2010; Vazquez-Roig, Andreu et al. 2010; Bierman and Sanchez 2010). He and Giusti (2011) explored the possibility of using mixed-mode sorbents for the isolation of anthocyanins from complex fruit and vegetable extracts. The combination of cation-exchange and hydrophobic interactions were utilized due to the ability of anthocyanins to acquire a positive charge in acidic conditions. Anthocyanin pigments have a greater affinity for the mixed-mode sorbent than that of the commonly used reversed-phase C 18 sorbent, as well as other established anthocyanin fractionation sorbents, resulting in less pigment loss during purification and isolates of superior purity. The increase in affinity results from the additional mechanism involving the cation-exchange interaction between the positively charged anthocyanin molecules (pk h ~ 2.8) and the negatively charged groups on the mixed-mode resin (He and Giusti 2011). He and Giusti (2011) successfully developed an innovative mixed-mode anthocyanin isolation method utilizing the cation-exchange and reversed-phase mechanisms of the Oasis MCX SPE cartridge. A wide variety of anthocyanins, from 12 edible sources, were purified in order to assess the overall ability of the method to purify anthocyanins. He and Giusti s method was found to be superior to the commonly used SPE methods regarding sorbent capacity, cost, simplicity, organic waste generation, anthocyanin recovery, and anthocyanin purity. Figure 1.12 depicts the sorbent structure of the Oasis MCX SPE cartridge utilized by He and Giusti (2011). Similar mixed-mode sorbents are also available from competing manufacturers. Figure 1.13 presents the structure of one of these sorbents which is active in Strata X-C mixed-mode SPE cartridges. 19

32 Figure 1.12 Oasis MCX sorbent: m-divinylbenzene-n-vinylpyrrolidone copolymers derivatized with sulfonic groups. Figure 1.13 Strata X-C sorbent: polar modified styrene-divinylbenzene. Availability of highly pure anthocyanin mixtures is of extreme importance to the food colorant and nutraceutical industries, as well as the research community. Due to the wide variety of naturally occurring anthocyanins and their differing stability to a multitude of factors there may only be a few possibilities for the coloration of a given food product. Additionally, the color produced by anthocyanins is influenced by a number of factors such as structural characteristics and environmental ph. Highly pure extracts will allow for a more precise determination of the proper natural colorant necessary to achieve the desired stable color. In addition to quickly and economically producing highly pure anthocyanin isolates, the method developed by He and Giusti (2011) is high throughput. This gives rise to the possibility of its application on a commercial scale, allowing for the production of large volumes of highpurity anthocyanin mixtures for use as colorants, as well as ingredients in nutraceuticals and functional foods. 20

33 1.4 PRESENT STUDY The objective of the present study was to explore the commercial applicability of the patent pending mixed-mode anthocyanin purification method. In order to achieve this goal the robustness of the method was assessed with previously examined and new pigment sources, its compatibility with another commercially available mixed-mode SPE cartridge was investigated, sorbent capacity was quantified, and cartridge longevity was analyzed. Anthocyanin isolate purity was also evaluated via molar absorptivity calculations, utilizing Lambert-Beer s law. High-purity anthocyanin mixtures minimize the interference from biologically active compounds and improve overall pigment stability, which may allow researchers to better validate the ability of anthocyanins to benefit human health. Pure anthocyanin standards are limited in number and extremely expensive, but this method promises to allow the vast majority of labs to create their own high-purity extracts at relatively low cost. The results from different labs will also be more comparable if the proposed method becomes the standard for anthocyanin purification by SPE. This research represents a necessary and exciting step in the evolution of anthocyanin purification, but more importantly in the evolution of the understanding of anthocyanins. 21

34 CHAPTER 2: MATERIALS AND METHODS 2.1 REAGENTS LC/MS grade Optima water, LC/MS grade Optima methanol, LC/MS grade Optima acetonitrile, high-performance liquid chromatography (HPLC) grade reagent alcohol (ethanol), reagent grade trifluoroacetic acid (TFA), certified American Chemical Society (ACS) grade formic acid, certified ACS plus grade ammonium hydroxide (NH 4 OH), and certified ACS grade sodium carbonate anhydrous powder were purchased from Fisher Scientific (Fair Lawn, NJ). Gallic acid was acquired from MP Biomedicals, LLC (Aurora, OH). Folin and Ciocalteau s phenol reagent was purchased from Sigma-Aldrich (St. Louis, MO). 2.2 ANTHOCYANIN SOURCES AND EXTRACT PREPARATION PLANT MATERIALS Fresh blackberries (product of Mexico), red table grapes, strawberries, blueberries, red sweet potatoes, and blue potatoes were purchased from a local Giant Eagle Market District grocery store. Fresh eggplant and red cabbage was purchased from a local Whole Foods Market SAMPLE PREPARATION A variety of anthocyanin-rich fruit and vegetable crude extracts were prepared using fresh produce. Approximately 50g of each plant material was quickly frozen in the presence of 22

35 liquid nitrogen (Rodriguez-Saona and Wrolstad 2001). The frozen produce was then placed into a chilled stainless steel blending container, and blended to a fine powder using a Waring commercial blender (Torrington, CT). Liquid nitrogen was added during blending in order to remove oxygen from the container and maintain a frozen, brittle plant material to achieve the desired fine powder. Powdering of the produce increases the surface area of the anthocyanin containing tissues, which allows for efficient and effective pigment extraction CHEMICAL EXTRACTION Two volumes (w/v) of 0.1% trifluoroacetic acid (TFA) acidified methanol was added to the finely powdered fruit and vegetable samples (He and Giusti 2011). The mixture was then homogenized using a Waring commercial blender and allowed to macerate for 1 hour (Rodriguez-Saona and Wrolstad 2001). Following maceration the slurry was filtered through Whatman number one filter paper via vacuum suction, utilizing a Buchner funnel. Reextractions were conducted, using methanol (0.1% TFA), until a faint-colored extract was obtained indicating that the majority of the pigment had been extracted from the plant material. The liquid extract was transferred to a boiling flask, and the methanol was evaporated using a Collegiate Büchi Rotavapor (Flawil, Switzerland) at 40 C under vacuum. Following methanol evaporation the crude extract was brought to a known volume with 0.1% TFA acidified LC/MS grade Optima water, and stored at or below 4 C. 23

36 2.3 SPE CARTRIDGES AND PURIFICATION METHODS MCX AND X-C SPE Oasis MCX mixed-mode SPE cartridges (6cc, 500mg sorbent, 53µm mean particle size) were acquired from Waters Corp. (Milford, MA). Strata X-C mixed-mode SPE cartridges (6cc, 500mg sorbent, 30µm mean particle size) were purchased from Phenomenex, Inc. (Torrance, CA). Prior to sample purification the mixed-mode SPE cartridges were conditioned with 2 volumes of methanol followed by 3 volumes of 0.1% TFA acidified water (He and Giusti 2011). Two volumes of aqueous crude extract were loaded onto the mixed-mode sorbent following conditioning. After loading the crude extract 2 volumes of water (0.1% TFA) were applied to the cartridge in order to wash the sample of any sugars and acids. Following the washing step, nonanthocyanin phenolics were removed by 2 volumes of methanol (0.1% TFA) and collected in a separate vial. Next, anthocyanins were eluted from the cartridge by 1 volume of methanol followed by 1 volume of water/methanol (40:60, v/v); both solvents contained 1% NH 4 OH. The anthocyanin fraction was eluted into a vial containing an aliquot of formic acid, bringing the basic eluate to a ph below 2, in order to avoid anthocyanin degradation C 18 SPE Sep-Pak Vac tc 18 SPE cartridges (6cc, 1g sorbent) were purchased from Waters Corp. (Milford, MA). Trace amounts of salt may have formed as a result of the neutralization of alkaline and acid during the elution of the anthocyanin fraction. The residual salt is generally not a concern for biological studies, but may be removed if desired. Removal of salt was accomplished by 24

37 loading the purified anthocyanin extract onto a C 18 cartridge (He and Giusti 2011). Prior to extract application the sorbent was conditioned with 2 volumes of methanol followed by 3 volumes of water (0.1% TFA). After the extract was loaded it was washed with 2 volumes of water (0.1% TFA) to remove the salt. Following washing the purified anthocyanin extract was eluted from the cartridge via 2 volumes of methanol (0.1% TFA) METHOD VALIDATION Crude extracts of red table grape, strawberry, blueberry, and red cabbage were prepared. Following extraction the crude extracts were purified, by triplicate, through Oasis MCX mixedmode SPE cartridges via the method proposed by He and Giusti (2011). The purified samples were analyzed by HPLC-PDA. All extracts were examined previously by He and Giusti (2011) using the proposed method and the Oasis MCX SPE cartridge. Purifications were conducted to analyze the robustness and validity of the proposed novel solid-phase extraction method. Crude extracts of blackberry, blue potato, and eggplant were prepared. Following extraction the crude extracts were purified, by triplicate, through Oasis MCX mixed-mode SPE cartridges via the method proposed by He and Giusti (2011). The purified samples were analyzed by HPLC-PDA. The purification method developed by He and Giusti (2011) had not been attempted previously with crude extracts of blackberry, blue potato, or eggplant CARTRIDGE CAPACITY Aqueous crude blackberry, grape, and strawberry extracts were prepared in order to study the sorbent holding capacity of the Oasis MCX and Strata X-C mixed-mode SPE cartridges. Four different quantities of each extract were loaded onto both mixed-mode cartridges. Quantities were selected based on visually observed leaching of anthocyanin pigment into the 25

38 phenolic fraction during purification. All phenolic fractions were retained for HPLC analysis. Anthocyanin fractions were combined and retained for HPLC and spectrophotometric analysis. Crude extracts were retained for spectrophotometric analysis. All samples were stored at or below 4 C CARTRIDGE LONGEVITY RESIN ENDURANCE Resin endurance was assessed using freshly prepared aqueous crude blackberry extract and new Oasis MCX and Strata X-C SPE cartridges. Two volumes of crude blackberry extract were loaded onto previously conditioned cartridges, by triplicate. The purification procedure proposed by He and Giusti (2011) was performed. Immediately following purification the cartridges were conditioned and an additional 2 volumes of crude blackberry extract were applied to each. Subsequent purification procedures were conducted, by triplicate. All anthocyanin fractions were retained for HPLC analysis. All cartridges were washed and stored in methanol for future extractions. Resin endurance was also assessed using aqueous crude blackberry extract stored frozen for two weeks and new Oasis MCX and Strata X-C SPE cartridges. The extract was thawed and 2 volumes were loaded onto the conditioned cartridges, by triplicate. Immediately following purification the cartridges were conditioned and an additional 2 volumes of crude blackberry extract was applied to each. Subsequent purification procedures were conducted, by triplicate. All anthocyanin fractions were retained for HPLC analysis. 26

39 RESIN EFFECTIVENESS FOLLOWING STORAGE Resin effectiveness following storage in methanol was assessed using aqueous crude blackberry extracts. The mixed-mode purification technique was conducted and then cartridges were stored in methanol for two weeks prior to reapplication. Crude blackberry extract was stored frozen for two weeks prior to the second purification. The extract was thawed and 2 volumes were applied to conditioned cartridges, by triplicate. Following purification all anthocyanin fractions were retained for HPLC analysis COMMERCIAL APPLICABILITY In order to assess the mixed-mode sorbents applicability in a processing environment, 20 purifications of aqueous crude blackberry extract (stored frozen >4 weeks) were run consecutively on previously unused Oasis MCX and Strata X-C SPE cartridges. The cartridges were conditioned prior to each purification procedure. 2.5mL of crude blackberry extract were loaded onto the sorbents for each of the twenty purifications, for a total of 50mL purified through each cartridge. Twenty consecutive purifications were conducted, by triplicate. All phenolic fractions, in addition to anthocyanin fractions, were retained for HPLC and spectrophotometric analysis. In all cases, samples were stored at or below 4 C. 2.4 SAMPLE ANALYSIS HPLC-PDA All phenolic and anthocyanin fractions were evaporated using a Collegiate Büchi Rotavapor (Flawil, Switzerland) at C, under vacuum, to remove organic solvents (He and 27

40 Giusti 2011). Following evaporation the samples were redissolved to a known volume in water (0.1% TFA), and filtered through a 0.45µm Whatman polypropylene syringe filter. Filtered samples were injected into HPLC vials, and analyzed using a Shimadzu HPLC-Photodiode array (PDA) system equipped with a SPD-M20A PDA detector and an MPC ClientPro 565 computer with Shimadzu LCMS Solution LCMS-2010EV software (Shimadzu Scientific Instruments, Inc., Columbia, MD). A Waters Corp. Symmetry C18 column (3.5µm, mm; Milford, MA) was utilized for compound separation. Additional chromatographic conditions included a flow rate of 0.8mL/min, mobile phase A: 4.5% formic acid in LC/MS grade water, mobile phase B: LC/MS grade acetonitrile, injection volume varied (20-200µL), and gradient: 0-5 minutes, 2-7% B; 5-20 minutes, 7-12% B; minutes, 12-17% B; minutes, 17-25% B; minutes, 25-2% B. After each run the column was equilibrated to initial conditions by running 2% B for 5 minutes. Anthocyanin elution was monitored at wavelengths from nm. Additionally, wavelengths from nm were monitored to collect data across the spectrum. Area under the curve (AUC) on the nm chromatogram was used to represent anthocyanin concentrations and AUC on the nm chromatogram was used to represent total phenolic concentrations for each sample (He and Giusti 2011). Sample purity (% purity) was determined by dividing the AUC of the anthocyanin peaks ( nm) by the AUC of the phenolic peaks ( nm) and multiplying by one hundred UV-VISIBLE SPECTROPHOTOMETRY TOTAL PHENOLICS A Shimadzu UV-2450 UV-visible Spectrophotometer equipped with a Dell Optiplex 745 computer and Shimadzu UVProbe 2.21 software was utilized for spectrophotometric analysis (Shimadzu Scientific Instruments, Inc., Columbia, MD). The microscale protocol for Folin- 28

41 Ciocalteau (FC) colorimetry was conducted in order to calculate total phenolics (Waterhouse 2002). Gallic acid calibration standards were prepared in concentrations of 50, 100, 250, and 500mg/L by dissolving 0.5g of gallic acid in 10mL of ethanol, diluting to 100mL with distilled water, and finally diluting 1,2,5, and 10mL to 100mL with distilled water. Standards were stored at 4 C, remaining potent for two weeks. A sodium carbonate solution was prepared by dissolving 100g of certified ACS grade sodium carbonate anhydrous powder in 400mL of distilled water. The solution was brought to a boil and immediately cooled. Following cooling, a small amount of sodium carbonate anhydrous powder was added to the solution which was then allowed to sit at room temperature for 24 hours. The solution was then filtered through Whatman number one filter paper via vacuum suction, utilizing a Buchner funnel. Following filtration, distilled water was added to bring the solution to 500mL. The sodium carbonate solution was stored indefinitely at ambient temperature. Twenty microliters of purified anthocyanin extracts, gallic acid calibration standards, and blank (LC/MS Optima water) were transferred into 1-cm path length, 2mL cuvettes, by triplicate. One Hundred microliters of FC reagent and 1.56mL of water were added to each of the cuvettes and mixed thoroughly by inversion. All solutions were allowed to incubate for 1-8 minutes. Following incubation, 300µL of the sodium carbonate solution was added to each of the cuvettes and mixed thoroughly by inversion. All solutions were allowed to incubate for 2 hours at room temperature. Following incubation, absorbance at 765nm was measured for each sample, gallic acid standard, and blank TOTAL MONOMERIC ANTHOCYANINS UV-visible spectrophotometry was also utilized for determining total monomeric anthocyanins via the ph-differential method (Giusti and Wrolstad 2001). A proper dilution factors (DF) was calculated for each sample, and the instrument was zeroed with distilled water at the visible maximum wavelength (λ vis-max ) of the most abundant anthocyanin in the sample and at 29

42 700nm. Two dilutions were prepared based on the predetermined dilution factor, one with 0.025M potassium chloride ph 1.0 buffer and the other with 0.4M sodium acetate ph 4.5 buffer. Each dilution was allowed to equilibrate for 15 minutes. Following equilibration the absorbance of each dilution was measured at the λ vis-max as well as at 700nm. The absorbance (A) was calculated using the equation: A = (A λ vis-max A 700 ) ph 1.0 (A λ vis-max A 700 ) ph 4.5. The total monomeric anthocyanin pigment concentration of the original sample was calculated using the formula: monomeric anthocyanin pigment (mg/liter) = (A MW DF 1000)/(ε 1), where MW is the molecular weight of the most abundant anthocyanin MOLAR ABSORPTIVITY In addition to calculating purity with HPLC-PDA data, purity was calculated based on molar absorptivity (ε), utilizing UV-visible spectrophotometry. Evaporatory flasks were thoroughly cleaned with soap and water, rinsed with reagent alcohol (ethanol), and placed in a desiccator to dry overnight. Dry flasks were weighed to the nearest milligram. Purified extracts were transferred into clean, weighed flasks and evaporated to dryness in a Collegiate Büchi Rotavapor at 40 C under vacuum. Flasks were dried with Kimwipes and transferred to a desiccator to dry overnight. Dried samples were weighed to the nearest milligram, taking into account the weight of the clean, dry flask. Tongs or Kimwipes were used to grasp flasks in order to guarantee cleanliness for accurate weighing. Purified dried anthocyanin extracts were dissolved in 10mL of LC/MS grade Optima methanol (Giusti, Rodriguez-Saona et al. 1999). An aliquot of each solution was diluted by 0.1% or 0.01% HCl acidified methanol and ph 1.0 buffer (0.2N KCl or 0.2M KCl/0.2M HCl (25:67, v/v)) (Giusti and Wrolstad 2001; Jordheim, Aaby et al. 2007). Dilutions were prepared in duplicates. Spectral data for each diluted sample was calculated using a 1-cm path length cell. Anthocyanin isolate purities ((experimental 30

43 conc./actual conc.)*100) were calculated according to Lambert-Beer s law using molecular mass, including the mass of a TFA counterion (Jordheim, Aaby et al. 2007). 2.5 STATISTICAL ANALYSIS Statistical analysis of percent purity, total phenolics, total monomeric anthocyanins, and FC colorimetry data was performed by one way analysis of variance (ANOVA) and/or simple regression analysis, with alpha levels of P>0.05, using Microsoft Office Excel with the Analysis ToolPak. 31

44 CHAPTER 3: RESULTS AND DISCUSSION 3.1 METHOD VALIDATION PIGMENT ISOLATION AND DETERMINATION OF PURITY Crude extracts of blueberry, red cabbage, red table grape, and strawberry (n=3) were purified via the proposed novel mixed-mode method utilizing, Oasis MCX mixed-mode SPE cartridges and analyzed by HPLC-PDA. Each of the four plant materials had been purified previously using the proposed mixed-mode technique. Chromatograms of each isolate are presented in Figure 3.01, portraying the variability in anthocyanin ( nm) compositions between the extracts. Purities calculated by % AUC were 99.5% (±0.5), 96.4% (±5.1), 92.2% (±4.3), and 91.0% (±0.6) for blueberry, red cabbage, red table grape, and strawberry, respectively. Inconsistencies between the nm chromatogram and the max-plot ( nm) represent impurities in the anthocyanin mixture. Figure 3.02 provides a visual representation of the calculation of purity by % AUC for a blueberry isolate. Calculated purities were compared to those previously determined by He and Giusti (2011), speaking to the robustness of the method (Figure 3.03). Exceptionally satisfactory purities were achieved for all extracts, reaffirming the ability of the method to produce high-purity anthocyanin mixtures from a variety of natural sources. 32

45 Figure 3.01 HPLC-PDA chromatograms of purified (A) red cabbage, (B) red table grape, (C) strawberry extracts observed at nm and nm. Figure 3.02 HPLC-PDA max-plot of purified blueberry extract, colored blue, area under anthocyanin peaks ( nm), filled in red. (AUC nm /AUC nm ) 100 = 99.9%. 33

46 Anthocyanin Isolate Purity (% AUC) present study He and Giusti blueberry red cabbage strawberry grape Commodity Figure 3.03 Comparison of the calculated purity (based on % AUC of HPLC-PDA chromatograms) of four commodities between studies. Present values are the mean ± SD (n=3). Crude extracts of blackberry, eggplant, and blue potato (n=3) were purified via the proposed novel mixed-mode method, utilizing Oasis MCX mixed-mode SPE cartridges. Figure 3.04 illustrates the difference between the crude and purified blue potato extract. The three plant materials had not been purified previously using the proposed mixed-mode technique. Anthocyanin fractions were analyzed by HPLC-PDA. A chromatogram of each isolate is presented in Figure Purities calculated by % AUC were 97.7% (±1.4), 93.5% (±2.7), and 74.2% (±7.1) for blackberry, eggplant, and blue potato, respectively. Calculated purities for blackberry and eggplant were similar (>90%) to those calculated previously for other commodities purified by the novel mixed-mode method (Figure 3.06). Isolation of blue potato anthocyanins proved to be somewhat difficult, providing an anthocyanin fraction of low purity, comparatively. This portion of the study was conducted prior to the implementation of a C 18 34

47 purification step following pigment isolation. The subsequent step was applied in order to remove salts produced through acid/base neutralization, but also serves as a second washing step for the removal of sugars and some acids. It is possible that the peaks prior to the ten minute mark on the blue potato chromatogram (Figure 3.05) represent aromatic acids, cleaved from the anthocyanins structure as a result of base saponification during anthocyanin elution under alkaline conditions, or degradation products produced by the enzymatic activity of polyphenol oxidase (PPO). Further assessment is needed in order to validate these possible explanations for the unachieved high-purity. Figure 3.04 HPLC-PDA max-plot chromatograms of (A) crude and (B) purified blue potato extract. Anthocyanins eluted at minutes. 35

48 Figure 3.05 HPLC-PDA chromatograms of purified (A) blackberry, (B) eggplant, and (C) blue potato extracts observed at nm and nm. 36

49 Anthocyanin Isolate Purity (% AUC) blackberry eggplant blue potato Commodity Figure 3.06 Isolate purity (based on % AUC of HPLC-PDA chromatograms) achieved for blackberry, eggplant, and blue potato crude extracts. Values are the mean ± SD (n=3). The innovative anthocyanin purification method showed promise in its ability to purify crude extracts prepared from a wide array of naturally pigmented materials. Brown pigmentation was observed during extraction of blue potato anthocyanins, most likely resulting from enzymatic activity by polyphenol oxidase (PPO). The activity of PPO may have hindered the ability of the method to produce an anthocyanin isolate of high purity; therefore, modifications should be made in order to inhibit PPO prior to pigment extraction. Increasing the acidity of the extracting solvent is recommended. 37

50 765 nm 3.2 CARTRIDGE CAPACITY TOTAL PHENOLICS The microscale protocol for FC colorimetry was followed in order to calculate the total phenolics (mg/l) present in blackberry, red table grape, and strawberry crude extracts, in association with the analysis of cartridge capacity. Figure 3.07 illustrates the linear trend line resulting from FC colorimetry calculations utilizing UV-visible spectrophotometry. Simple regression analysis was conducted indicating the significance (P<0.01) of the slope. The absorbances (n=2) of crude extracts at 765nm were plugged into the equation of the trend line in order to calculate the concentration (mg/l) of total phenolics (Table 3.01) as gallic acid equivalents (GAE) y = x R² = Concentration GAE (mg/l) Figure 3.07 FC colorimetry: GAE vs. absorbance at 765nm. Values are the mean ± SD (n=3). 38

51 3.2.2 TOTAL MONOMERIC ANTHOCYANINS The ph-differential method was followed in order to calculate the total monomeric anthocyanin concentration (mg/l) of blackberry, red table grape, and strawberry crude extracts (n=2), in association with the study of cartridge capacity. Table 3.01 provides the total monomeric anthocyanins calculated for each crude extract. For the blackberry and strawberry crude extracts, anthocyanins accounted for approximately 30% of all phenolic compounds (Table 3.01), whereas the red table grape anthocyanins only accounted for roughly 11% of the extracts total phenolics. This difference may have resulted from the fact that the majority of fresh blackberry and strawberry tissues, used in this study, were pigmented throughout, whereas only the tissues of the skin of the fresh red table grapes were pigmented. It is probable that the percentage of total phenolics consisting of anthocyanins would have been much higher had only the skins of the red table grapes been utilized. Extract Blackberry Grape Strawberry Total Phenolics (mg/l) Total Anthocyanins (mg/l) Table 3.01 Total phenolic and monomeric anthocyanins concentrations of three crude extracts SORBENT HOLDING CAPACITY Sorbent holding capacity was analyzed following the determination of total phenolics and total monomeric anthocyanins in the crude blackberry, red table grape, and strawberry extracts. Four quantities of each crude extract were loaded onto fresh Oasis MCX and Strata X-C mixed-mode SPE cartridges. Extract quantities loaded onto the sorbents were increased until the 39

52 visually observed occurrence of pigment leaching in the phenolic fraction during purification. The range of extracts applied to the mixed-mode resins are provided in Table The amount of anthocyanins (mg) added was fairly consistent across extracts, whereas the amount of phenolics applied was much greater for the red table grape extract. As previously stated, this is likely directly related to the lower percentage of anthocyanins, in relation to total phenolics, in the red table grape sample. Crude extract Blackberry Grape Strawberry Phenolics (mg) Anthocyanins (mg) Table 3.02 Quantity of phenolics and anthocyanins of three crude extracts loaded onto mixedmode sorbents. Ranges from start to point where pigment was observed in the phenolic fraction. Various quantities of crude blackberry, red table grape, and strawberry extracts were purified via the proposed innovative mixed-mode anthocyanin purification technique. During purification the phenolic fractions were retained for HPLC-PDA analysis. Figure 3.08 provides a visual representation of the calculation of anthocyanin loss in the phenolic fraction by % AUC, following the loading of 0.71mg of anthocyanins/2.42mg of phenolics. The vast majority of anthocyanin molecules were retained by the sorbent, as a small proportion eluted with the phenolic fraction (5.5% of the total phenolics in the fraction). 40

53 Figure 3.08 Calculation of pigment loss in phenolic fraction. HPLC-PDA chromatograms of blackberry phenolic fraction following loading of 0.71mg of anthocyanins. Anthocyanins observed at (A) nm and overall composition observed at (B) nm. (AUC A /AUC B ) 100 = 5.5%. Percent anthocyanins in the phenolic fraction (n=3) were calculated as % AUC for blackberry, red table grape, and strawberry extracts. Percent AUC of anthocyanin peaks in the phenolic fraction were plotted against loaded anthocyanins (mg) in order to determine if pigment loss/sorbent capacity was linearly related to the amount of anthocyanins applied to the sorbent (Figures ). To assess sorbent holding capacity, acceptable anthocyanin peak AUC was established as 5% anthocyanins in the phenolic fraction. Linear trend lines were created for all data sets in order to assist in the determination of the limit of the sorbent s anthocyanin binding capacity. 41

54 Grape Anthocyanins Lost in Phenolic Fraction (% AUC) Blackberry Anthocyanins Lost in Phenolic Fraction (% AUC) Oasis MCX Strata X-C y = x R² = y = x R² = Loaded Anthocyanins (mg) Figure 3.09 Percentage of blackberry anthocyanins in the phenolic fraction. Values are the mean ± SD (n=3) Oasis MCX Strata X-C y = x R² = y = x R² = Loaded Anthocyanins (mg) Figure 3.10 Percentage of red grape anthocyanins in the phenolic fraction. Values are the mean ± SD (n=3). 42

55 Strawberry Anthocyanins Lost in Phenolic Fraction (% AUC) Oasis MCX Strata X-C y = x R² = y = x R² = Loaded Anthocyanins (mg) Figure 3.11 Percentage of strawberry anthocyanins in the phenolic fraction. Values are the mean ± SD (n=3). Data regarding the anthocyanin holding capacity of the mixed-mode sorbents is presented in Table Neither of the mixed mode sorbents significantly outperformed (P>0.05) the other in terms of anthocyanin holding capacity. Similarly, there was no statistically significant difference (P>0.05) in the anthocyanin holding capacity of the sorbents between the different crude extracts. This is extremely promising, revealing that the sorbents perform similarly across a wide range of pigment sources and with a variety of anthocyanin structures, as blackberries contain mostly cyanidin, red grapes contain mostly malvidin, and strawberries contain mostly pelargonidin (U.S.D.A. Agricultural Research Service 2007). These findings uphold the indication of the proposed method s applicability with all anthocyanins, resulting from the ability to become protonated at low ph. The similarity in performance between the mixed-mode sorbents may indicate that the principle of mixed-mode separation is applicable with multiple commercially available SPE cartridges. 43

56 Crude Extract Blackberry Grape Strawberry Total Phenolics (mg/l) Total Anthocyanins (mg/l) Oasis MCX Strata X-C Oasis MCX Strata X-C Oasis MCX Strata X-C Phenolics (mg) Capacity, Anthocyanins (mg) Table 3.03 Mixed-mode sorbent anthocyanin holding capacity in relation to total phenolics and monomeric anthocyanins of three crude extracts. The only statistically significant difference (P<0.001) in the cartridge capacity data was seen in the quantity of phenolics loaded to achieve the sorbent s anthocyanin holding capacity. A much higher quantity of red table grape phenolics were applied to the sorbents compared to that of blackberry and strawberry. This resulted from the previously stated fact that anthocyanins only accounted for approximately 11% of the total phenolics of the red table grape extract, whereas anthocyanins accounted for roughly 30% of the total phenolics of the blackberry and strawberry extracts. The amount of non-anthocyanin phenolics applied to the mixed-mode resins had limited to no effect on anthocyanin binding capacity, as the ability of anthocyanins to adhere to the solid phase is likely directly associated with the number and availability of cation-exchange binding sites (sulfonic groups). 44

57 Anthocyanin Isolate Purity (% AUC) 3.3 CARTRIDGE LONGEVITY RESIN ENDURANCE Mixed-mode resin endurance was studied using freshly prepared crude blackberry extract (n=3) and new Oasis MCX and Strata X-C SPE cartridges. Two successive purification procedures were conducted on each mixed-mode sorbent. Isolates were analyzed by HPLC-PDA to determine purity based on % AUC. High-purity (>96.5%) was achieved for all purified blackberry anthocyanin mixtures. There was no statistical difference (P>0.05) in the purity achieved by either cartridge or resulting from a subsequent purification procedure. Figure 3.12 offers the isolate purities achieved, resulting from the cartridge employed as well as the purification run number st Purification 2nd Purification Oasis MCX SPE Cartridge Strata X-C Figure 3.12 Blackberry anthocyanin isolate purity. Successive purifications with fresh extract and fresh mixed-mode sorbents. 45

58 Anthocyanin Isolate Purity (% AUC) The resin endurance of the cartridges was also assessed using crude blackberry extract that was stored frozen for two weeks in combination with new Oasis MCX and Strata X-C SPE cartridges (n=3). Two successive purification procedures were conducted on each mixedmode sorbent. Isolates were analyzed by HPLC-PDA to determine purity based on % AUC. High-purity (>97.4%) was achieved for all purified blackberry anthocyanin mixtures (Figure 3.13). There was no statistical difference (P>0.05) in the purity achieved by either cartridge or resulting from a subsequent purification procedure. Furthermore, frozen storage and thawing of the crude extract had no effect (P>0.05) on final isolate purity st Purification 2nd Purification Oasis MCX SPE Cartridge Strata X-C Figure 3.13 Blackberry anthocyanin isolate purity. Successive purifications with extract stored frozen 2 weeks and fresh mixed-mode sorbents. Similarity in isolate purity between cartridges further suggests the compatibility of the mixed-mode anthocyanin purification method with multiple cation-exchange/reversed-phase resins. The ability of the sorbents to maintain functionality across multiple uses is promising for eventual application in a production type setting. 46

59 Anthocyanin Isolate Purity (% AUC) RESIN EFFECTIVENESS FOLLOWING STORAGE In addition to assessing the endurance of the resin across multiple purification procedures, resin effectiveness following storage in methanol was evaluated. Resin effectiveness was studied using freshly prepared crude blackberry extract and new Oasis MCX and Strata X-C SPE cartridges (n=3). A single purification procedure was conducted on each mixed-mode sorbent prior to storage in methanol and following two weeks of storage. Isolates were analyzed by HPLC-PDA to determine purity based on % AUC. High-purity (>97.0%) was achieved for all isolated blackberry anthocyanin mixtures (Figure 3.14). There was no statistical difference (P>0.05) in the purity achieved by either cartridge or resulting from a subsequent purification procedure. In other words, the mixed-mode sorbents maintained their effectiveness following two weeks of storage in methanol st Purification 2nd Purification Oasis MCX SPE Cartridge Strata X-C Figure 3.14 Blackberry anthocyanin isolate purity. Successive purifications with extract stored frozen 2 weeks and used mixed-mode sorbents stored in MeOH 2 weeks. 47