Isolation of B-Carotene from Carrot.pdf

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Karlstads universitet 651 88 Karlstad Tfn 054-700 10 00 Fax 054-700 14 60 [email protected] www.kau.se Faculty of Technology and Science Chemistry Annica Wingqvist Extraction, Isolation and Purification of β-carotene Analytical Chemistry Diploma Work Date/Term: VT 2011/HT 2011 Supervisor: Jörgen Samuelsson Torgny Fornstedt Examiner: Torgny Fornstedt

Transcript of Isolation of B-Carotene from Carrot.pdf

  • Karlstads universitet 651 88 Karlstad Tfn 054-700 10 00 Fax 054-700 14 60

    [email protected] www.kau.se

    Faculty of Technology and Science

    Chemistry

    Annica Wingqvist

    Extraction, Isolation and Purification

    of -carotene

    Analytical Chemistry

    Diploma Work

    Date/Term: VT 2011/HT 2011

    Supervisor: Jrgen Samuelsson

    Torgny Fornstedt

    Examiner: Torgny Fornstedt

  • Abstract

    With focus on purification and isolation, the fat soluble, highly conjugated hydrocarbon -carotene

    was investigated. The aim originated from a research project whose objective is an attempt to take

    better advantage of the planets resources, doing this particularly by refining by-products from the

    industry, which e.g. include -carotene. -carotene, known for its provitamin A activity and potential

    disease suppression, as well as usage as a colorant, is a sought compound. This high-value compound

    in this study found in carrot extract originating from pressurized fluid extraction (PFE) have been

    detected and purified by high performance (pressure) liquid chromatography (HPLC). The extract

    should prior to usage exhibit profitable and environmental friendly -carotene concentration. By solid

    phase extraction (SPE) four times higher concentration than the original one was achieved.

    Competitive extraction methods were developed and compared with PFE, by amount extracted in

    correlation to time consumed particularly reflux boiling proved to be a good option. Owing to

    reactivity -carotenes instability and degradation are stated in other studies, in this study this is on

    certain points rejected, e.g. heat instability and the usefulness of an antioxidant (butylated

    hydroxytoluene (BHT)). The later requires nevertheless more long term studies to actually confirm the

    pointlessness in using BHT. Further investigation and evaluation of the SPE method and the extraction

    methods will also be necessary prior to scale-up of the process for industrial application.

  • Sammanfattning

    Med fokus p rening och isolering har det fettlsliga, starkt konjugerade kolvtet -karoten utforskats.

    Ursprunget till studien r en del i ett forskningsprojekt vars syfte r att bttre ta vara p jordens

    resurser, framfrallt genom frdling av restprodukter ifrn industrin vilket inkludera bl a. -karoten.

    -karoten uppvisar provitamin A aktivitet, r potentiellt sjukdomsfrebyggande samt anvnds som

    frgmne inom olika delar av industrin, de aktuella anvndningsomrden har resulterat i att -karoten

    r en eftertraktad frening. Kllan till -karoten fr studien var frst och frmst morotsextrakt

    utvunnet genom trycksatt lsningsmedelsextraktion (PFE), -karotenen detekterades och renades

    genom vtskekromatografi (HPLC). Fr att utgra ett lnsamt och miljvnligt alternativ br extraktet

    uppvisa en tillrckligt hg -karotenkoncentration. Genom fastfasextraktion (SPE) erhlls fyra gnger

    hgre koncentration i frhllande till ursprungslsningen. Under studien utvecklades ocks

    jmfrande extraktionsmetoder, en metod baserad p terloppskokning uppvisade, genom mngden

    extraherad -karoten i korrelation till frbrukad tid, konkurrenskraft gentemot PFE. Olika studier har

    pvisat att -karoten vid hantering r instabil och ltt bryts ned p grund av att den har ltt att reagera,

    faktum som tillbakavisa p flera punkter i den hr studien s som -karotens vrmeinstabilitet och

    behovet att tillstta en antioxidant (butylerad hydroxytoluen (BHT)). Det senare erfodrar dock mer

    lngsiktiga studier fr att eventuellt bekrfta det verfldiga i att anvnda BHT. Vidare forskning och

    utvrdering av SPE-metoden och extraktionsmetoderna r ocks ndvndigt fre en uppskalning av

    processen fr industriella tillmpningar.

  • Abbreviations

    BHT: Butylated Hydroxytoluene

    CV: Coefficient of Variation

    DAD: Diode Array Detector

    EtOH: Ethanol

    HPLC: High Performance (Pressure) Liquid Chromatography

    LOD: Limit of detection

    LOQ: Limit of quantification

    MeOH: Methanol

    MTBE: Methyl Tert-Butyl Ether

    PFE: Pressurized Fluid Extraction

    SPE: Solid Phase Extraction

    SuReTech: Sustainable Resource Technology

    UV/VIS: Ultraviolet/Visible

  • Table of Contents 1. Introduction ......................................................................................................................................... 9

    1.1 Background ................................................................................................................................... 9

    1.2 Carotenoids .................................................................................................................................. 10

    1.2.1 -carotene ............................................................................................................................. 10

    1.3 Aim of study ................................................................................................................................ 11

    1.4 Theoretical background ............................................................................................................... 12

    1.4.1 High Performance Liquid Chromatography (HPLC) ........................................................... 12

    1.4.2 Extraction methods ............................................................................................................... 14

    1.4.2.1 Traditional boiling in ethanol ........................................................................................ 14

    1.4.2.2 Reflux boiling ................................................................................................................ 14

    1.4.2.3 Soxhlet ........................................................................................................................... 15

    1.4.2.4 Pressurized Fluid Extraction (PFE) ............................................................................... 15

    1.4.2.5 Solid phase extraction (SPE) ......................................................................................... 16

    2. Material and methods ........................................................................................................................ 17

    2.1 Chemicals .................................................................................................................................... 17

    2.2 Samples ....................................................................................................................................... 17

    2.3 Instrumentation ............................................................................................................................ 17

    2.4 Chromatographic condition ......................................................................................................... 17

    2.5 Standard preparation .................................................................................................................... 17

    2.6 Sample preparation ...................................................................................................................... 18

    2.7 Extraction procedures .................................................................................................................. 18

    2.7.1 Carrot extractions in this study ............................................................................................. 18

    2.7.2 Pressurized Fluid Extraction (PFE) (reference method) ....................................................... 18

    2.7.3 Solid phase extraction (SPE) ................................................................................................ 18

    2.8 Solubility test ............................................................................................................................... 18

    2.9 BHT studies ................................................................................................................................. 19

    2.10 Heat stability.............................................................................................................................. 19

    2.11 Analytical tools .......................................................................................................................... 19

    3. Results and discussion ....................................................................................................................... 21

    3.1 HPLC method evaluation ............................................................................................................ 21

    3.1.1 Separation and detection....................................................................................................... 21

    3.1.2 Linearity and Precision ......................................................................................................... 21

    3.1.3 Limit of detection and quantification ................................................................................... 22

    3.2 Solubility of -carotene ............................................................................................................... 23

    3.3 Stability of -carotene ................................................................................................................. 23

    3.3.1 BHT-studies .......................................................................................................................... 23

    3.3.2 Heat stability ......................................................................................................................... 24

    3.4 Increasing the concentration of -carotene .................................................................................. 25

  • 3.4.1 Solid Phase Extraction .......................................................................................................... 25

    3.5 Extraction of -carotene .............................................................................................................. 29

    4. Conclusions ....................................................................................................................................... 33

    5. Acknowledgements ........................................................................................................................... 35

    6. References ......................................................................................................................................... 37

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    1. Introduction

    1.1 Background

    When we use the resources of the earth it would be for the common good to take advantage of all

    constituents. Today the agriculture and the forestry are the cause of much waste. E.g. unapproved

    vegetables and other byproducts become decomposed and combusted to natural gas, directly

    combusted or used as animal feed. Valuable compounds are thrown away in these processes,

    economically profitable compounds that can be used in different lines of business such as health,

    grocery and cosmetics.

    Groups of researcher within Sustainable Resource Technology (SuReTech) are aimed to take care

    of these for industry interesting compounds prior to combustion, special focus is directed towards

    polyphenolic antioxidative molecules and fat soluble vitamins. The sources for these compounds are

    e.g. birch bark, carrots, onion and apple waste.

    In order to do this the researcher will go through all the steps in the chemical process, the

    extraction, modifying and isolation of the valuable compounds, having a holistic approach in mind,

    certain research groups are also focusing in the potential of integrate lifecycle assessment and a

    socioeconomic perspective (Figure 1) [1].

    Figure 1 Production line from industry to purified high-value compound.

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    1.2 Carotenoids

    From the agricultural industry carrots not meeting the market requirements give rise to much waste.

    By chance they contain the valuable class carotenoids, fat soluble compounds which are of great

    interest to the market [2].

    The carotenoids are built up as large conjugated hydrocarbon skeletons (Figure 2) [3, 4].

    Figure 2 Isoprene, five carbon building blocks, precursor in the synthetic route resulting in the carotenoid

    lycopene, which in turn is the precursor of many other carotenoids.

    The conjugated double bond system is the foundation to carotenoids special properties, makes them

    easy reacting due to their highly energetic delocalized -electrons, which requires little energy for

    excitation. Little energy and hence longer wavelength resulting in absorption in the UV/VIS region

    and are the reason that carotenoids appears colored, e.g. -carotene absorbs light at 450 nm and turns

    out orange [4].

    Carotenoids are for better and worse reactive, unfortunately causing problems to handle them. When

    working with carotenoids earlier researcher have consider the influence of heat, light, oxygen, acid and

    alkaline conditions [3, 5]. Different solutions to overcome the problem have been used; working away

    from daylight, work under nitrogen, storage in the freezer and addition of different antioxidants,

    particularly butylated hydroxytoluene (BHT) [5, 6, 7, 8].

    1.2.1 -carotene

    -carotene is one of the major carotenoids (Figure 3), and is one among them who long have been

    known to exhibit provitamin A activity [3, 9]. Carotenoids also exhibit antioxidative properties [10,

    11] due to their appearance, leading to that they nowadays are thoroughly investigated to reduce the

    risk of different diseases owing to the antioxidant effect [12, 13]. Studies of this correlation have also

    become of great interest since consumption of fruits and vegetables containing these compounds

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    eventually have led to reduced risk to develop for example cancer [14, 15]. Many studies have

    investigated and correlate the carotenoids, particularly -carotene, and their prohibitive effect on

    diseases including cancer [14, 16, 17, 18, 19], photosensitivity disorders [10, 20], cardiovascular

    diseases [16, 21], age-related ones [22, 23, 24], but recent studies, often more long-term are a

    sometimes contradictory [24, 25, 26] and some even describes a negative correlation between -

    carotene together with lung cancer and cardiovascular disease in smokers [27, 28], the investigations

    will be continued.

    Figure 3 Molecular structure of -carotene.

    Besides their health related role another use for the carotenoids are as food, cosmetic and personal care

    products colorant [29, 30], where in the later one the possible health benefit once again is taken into

    account [31].

    Regardless of carotenoids real effect or not they and particularly -carotene are a part of a growing

    market, the majority is produced chemically [2, 30].

    1.3 Aim of study

    Much analytical work has as mentioned been made on carotenoids. In addition to investigate their role

    as health promoter, many other studies have focused on extracting and investigating the constituents of

    different origins, by different analytical methods [32, 33, 34, 35, 36, 37]. However not many attempts

    have been made to use the -carotene and further scale-up the process.

    The main aim of the study was to purify and concentrate -carotene from carrot extract made by

    pressurized fluid extraction (PFE). For comparison to the PFE from the SuReTech project,

    developments of new extraction method were made. Another minor study performed was to

    investigate -carotenes stability.

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    1.4 Theoretical background

    1.4.1 High Performance Liquid Chromatography (HPLC)

    High Performance (Pressure) Liquid Chromatography (HPLC) was used as analysis method.

    The principle behind HPLC and the chromatographic method in general is separation between

    different components in a sample. This separation is achieved according to a number of equilibrium

    stages where the injected samples components interact by partition or adsorption between the

    stationary and the mobile phase during movement through the system.

    Depending on chemical and physical properties components in the sample exhibit different affinity

    for the mobile and the stationary phase and migrate through the column at different rate. A more

    retarded component has an equilibrium ratio favoring the stationary phase more [15]. The resolution,

    RS, is a measure of the separation efficiency which depends on how far apart and how broad the peaks

    showed in a chromatogram (a graph representing the detector response as a function of time (Figure

    4)) are (Equation 1) [38].

    (1)

    tR is the separation in time between peaks and wav is the average width of the two peaks, where w is

    the peaks width at the baseline. An alternative way to represent it is also shown in Equation 1, w1/2av is

    the width of the half-height of the peak (Figure 4), for calculation usually easier to measure,

    particularly because the peaks seldom exhibit such a good separation as in Figure 4.

    Figure 4 Schematic picture of a chromatogram presenting the resolution parameters.

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    A good separation between components exhibit a resolution > 1.5 [38].

    The band broadening contributing to the resolution is affected by A multiple paths, B longitudinal

    diffusion, C mass transfer of solute between stationary and mobile phase (these are constant for a

    given stationary phase and column) and uX the linear flow rate according to van Deemters equation

    (Equation 2) [38]:

    (2)

    Decreasing H, the plate height, gives narrower peaks and improved separation of components,

    equivalent to increasing efficiency. A smaller plate height is equal to a larger N, number of theoretical

    plates, which increase column efficiency (Equation 3) [38].

    (3)

    L is the column length.

    A rapid separation progress without affecting the resolution is sought for in chromatography

    particularly in the industrial production.

    As the method name HPLC indicates high pressure force the mobile phase through the column

    containing the stationary phase [38]. The most commonly used scheme is reversed phase

    chromatography [38], where the stationary phase is non-polar or weakly polar and the mobile phase is

    more polar. These conditions result in that non-polar solvent are retained in the stationary phase.

    Modifying the mobile phase to become more non-polar elutes also the most strongly retained solutes.

    The solutes reaching one by one the detector and are depicted in the chromatogram as mentioned

    above and then possibly collected for further use. A schematic picture of a HPLC system is shown in

    Figure 5.

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    Figure 5 HPLC system, octadecyl (C18) is the stationary phase in the column.

    1.4.2 Extraction methods

    The extraction process implies a separation of a substance from a matrix.

    1.4.2.1 Traditional boiling in ethanol

    By boiling for example a precipitate in a solvent by one or several sessions the sought substance will

    be extracted. Filtration [39] is required after the extractions before analyzing the extract further.

    1.4.2.2 Reflux boiling

    The sample and the solvent are placed in a round-bottom flask equipped with a condenser (Figure 6).

    The mixture is heated to reflux for a predetermined time [39]. Sample will possibly be taken during

    the time of the session to analyze the reaction progress, eventually all the solute is extracted from the

    matrix without too much solvent consumption.

    Figure 6 Reflux equipment.

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    1.4.2.3 Soxhlet

    The apparatus is a little more advance than the reflux boiling equipment. Between the condenser and

    the round bottom flask containing the solvent a soxhlet extractor containing a chamber carrying a

    thimble, made of thick filter paper, packed with the solid to be extracted, is placed (Figure 7). The

    solvent is heated to reflux, the vapour travel through the passage of the extractor and the condensate

    drips back down into the solid material. The solvent fills up the chamber while the material is

    extracted from the solid. When the solvent volume has almost filled the chamber, the solvents

    containing the extract flows back into the round bottom flask through a siphon tube. The process is

    repeated for a number of cycles and the extract is accumulated in the round bottom flask. Running the

    extraction for a prolonged period may extract material that is only slightly soluble in the solvent [39].

    Figure 7 Soxhlet apparatus.

    1.4.2.4 Pressurized Fluid Extraction (PFE) (the method of the Lund participants)

    The pressure fluid extraction technique takes advantage of high pressure and controlled temperature to

    achieve shortened extraction time and less solvent consumption. By the high pressure the solvent that

    would otherwise boil in the atmosphere can be used, it also a making the extraction more efficient by

    forcing the solvent into the matrix. According to the low amount of solvent this will be the greenest

    alternative (even though not the most efficient alternative). The procedure followed is: The solvent

    arrive into the extraction cell, heating causing the solvent to expand. After the apparatuses reaching a

    given set value the solvent is forced into the sample to be extracted, this will be done during a

    predetermined time and a given number of cycles each in which the solvent are replaced by fresh [40].

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    1.4.2.5 Solid phase extraction (SPE)

    The solvent containing the extracted material are applied to a column carrying a small volume of

    stationary phase. The solutes interact and absorb to the stationary phase depending on the solutes and

    the chosen phase, then impurities are washed out and finally the substance of interest is eluted by an

    small volume of an appropriate solvent, this time preferably one with a better solving properties

    toward the solute (Figure 8) [38]. By this approach most of the unnecessary sample matrix are washed

    away which results in simpler analysis or as is in this study also a concentration of the solved extract.

    Figure 8 Schematic picture of SPE.

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    2. Material and methods

    2.1 Chemicals

    Methanol and methyl tert-butyl ether (MTBE) both HPLC grade qualities were obtained from Fisher

    Scientific (Loughborough, UK). -carotene (purum 97% (UV)), butylated hydroxytoluene (BHT) (

    99%) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Ethanol (95%) was purchased

    from Solveco AB (Rosersberg, Sweden).

    2.2 Samples

    Carrots used were purchased at the local supermarket, cultivar Napoli. The carrots were prepared by

    running twice through a juice extractor and then use the solid material for further -carotene

    extraction.

    2.3 Instrumentation

    The apparatus for the chromatographic purpose was a high performance liquid chromatography 1200

    series system (Agilent Technologies, Waldbronn, Germany) equipped with an 900 L (200 bar) auto

    sampler and a diode array detector. The system was controlled and the data handled by Agilent

    ChemStation for LC 3D Systems (Agilent Technologies, Waldbronn, Germany). The analytical

    column was a C18 (150 46 mm, 5 m particles) Kromasil (Eka Chemicals AB, Bohus, Sweden)

    interconnected with a filter PEEK end fitting 0.5 m Pk10 (Thermo Scientific, Waltham, MA, USA).

    Solid Phase Extraction was performed on 50 mg Isolute solid phase extraction column

    (International Sorbent Technology Ltd., Mid-Glamorgan, UK). The juice extractor was a Philips HR

    1858 (Philips AB, Stockholm, Sweden).

    2.4 Chromatographic condition

    The developed HPLC analytical method for separation of -carotene was achieved isocratically with

    the C18 column kept under constant temperature (22.9C set at 21C) inside a condenser and a flow

    rate set at 2.0 ml/min. The mobile phase used was a mixture of Methanol and MTBE (60:40 v/v). The

    detection of -carotene was obtained at 450 nm.

    2.5 Standard preparation

    A stock solution with the final concentration of 50 mg/L was prepared by dissolving -carotene in

    MTBE, filtrate and then add methanol. A calibration curve in the range 0-10 mg/L was set up, where

    the sample was diluted in the mobile phase. This procedure was done daily according to the possible

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    instability of the -carotene solution. Other special precautions were taken by keeping the samples

    away from light and store them in the freezer.

    2.6 Sample preparation

    The sample analyzed directly after the carrot extraction was dissolved in 40:60 EtOH:MTBE.

    Standards and samples prepared by SPE were dissolved in 40:60 MeOH:MTBE.

    2.7 Extraction procedures

    2.7.1 Carrot extractions in this study

    o Traditional boiling in ethanol: The solid carrot starting material (~20 g) was boiled in 2100

    mL ethanol for 230 minutes, the ethanol solution was filtered of through a juice- and a coffee

    filter between the sessions.

    o Reflux boiling: In the second method the solid carrot starting material (20-50 g) was run

    through 30 minutes in 200 mL ethanol with sample taken and evaluated every fifth minute.

    o Soxhlet was used for five cycles of reflux boiling, between each cycle a sample was taken for

    concentration determination. The amount of carrot was approximately 20 g and the volume

    ethanol 200 mL.

    2.7.2 Pressurized Fluid Extraction (PFE) (reference method)

    According to description 10 g carrot (different cultivar in contrast to this study) where homogenized

    by a food processor and dried by grinding the carrot with hydromatrix in a mortar. The dried carrot

    was then put into an extraction vial. The set parameters were pressure (50 bars); preheat (0 min); heat-

    up time (5 min); flush volume (60%) and purge time (30 sec). The temperature was set at 60C,

    extraction time was 5 cycles, 2 min each.

    2.7.3 Solid phase extraction (SPE)

    A 50 mg C18 SPE column was used. The SPE-procedure was as follows: precondition by adding 1-3

    mL methanol, a known amount of sample was sat on the column, methanol (21 mL) was used to

    wash the column and finally the -carotene sample was eluted by methyl tert-butyl ether (MTBE).

    2.8 Solubility test

    -carotene was added to the solvent until a saturated solution was achieved. The solution was filtered

    to remove unsolved carotene and then measurement was made.

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    2.9 BHT studies

    Several studies have used butylated hydroxytoluene (BHT) as antioxidant. To examine its usefulness

    the influence of BHT on -carotene solutions were studied during several days, under various

    conditions:

    1. sample kept in the autosampler (no freezing, but kept away from light)

    2. sample stored in the freezer between the measurements

    3. sample stored in the daylight between the measurements (no freezing)

    4. sample kept in the autosampler during the day, but stored in the freezer over night

    Each of the four categories includes two samples, one with and one without BHT.

    2.10 Heat stability

    -carotene is seen as heat unstable, to investigate if it would affect the extraction a heat stability test

    was performed. 25 mg -carotene was dissolved in 250 mL ethanol, filtered and then boiled,

    temperature constant at 70C. Every fifth minute a sample from the boiling was analyzed.

    2.11 Analytical tools

    Statistical analysis was performed in Excel (Microsoft Corporation, Redmond, WA, USA).

    Significance level was 0.05.

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    3. Results and discussion

    3.1 HPLC method evaluation

    The method was developed after considering various articles dealing with separation of carotenoids

    [33, 34, 35, 41, 42, 43, 44], which exhibited a wide variety in approaches. The condition chosen here

    was influenced by them but adapted to prevailing circumstances, e.g. the environmental aspect.

    3.1.1 Separation and detection

    In the determination method developed a -carotene peak was detected between only 2-3 minutes. The

    sample was further observed at various wavelengths thus detect impurities with absorbance maximum

    different from 450 nm, no considerable disturbances in the -carotene region were observed. Peaks

    indicating other substance (e.g. impurities) was eluted and thus detected earlier in the chromatogram

    (Figure 9). t0 was determined by potassium nitrate at 0.798 min.

    Figure 9 Schematic picture of a chromatogram where -carotene is clearly identified.

    3.1.2 Linearity and Precision

    The developed analytical method showed linearity for -carotene between 0-10 mg/L. When repeated

    measurement was made on the same solution (n = 5) no notable difference was achieved, CV = 0.30-

    0.60 %. The repeatability between the three standards (10 mg/L, same stock solution) the coefficient

    of variation was 0.79 %.

    Later the same day the first sample exhibit a CV = 1.18% (n = 9, five and four measurement each

    session) in -carotene concentration, on to the following day the sample showed no differences CV =

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    0.02% (n = 9, four and five measurements for each session). Significant differences were then detected

    when the sample was stored for longer periods, thereby new standards was prepared each day

    (between the measurements the sample was stored in the freezer).

    The standard solution freshly prepared each day became the basis for the (reproducibility)/inter-day

    repeatability measurements which exhibit a coefficient of variation 2.50 % (in concentration) for five

    different days.

    3.1.3 Limit of detection and quantification

    The limit of detection (LOD) 3 the noise and the limit of quantification (LOQ) 10 the noise was

    determined at 0.22 and 0.73 mg/L respectively (Table 1). The noise was determined by the highest

    detected signal in a chromatogram without any sample injected.

    Table 1 Evaluation parameters of -carotene.

    Range of linearity (mg/L) 0-10

    Repeated measurements, one

    standard (n = 5)

    0.30-0.60 %

    Repeatability of standards (n = 3) 0.79 %

    Inter-day repeatability (n = 5) 2.50 %

    LOD (mg/L) 0.22

    LOQ (mg/L) 0.73

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    3.2 Solubility of -carotene

    Solubility test for -carotene in MTBE and the solvent MeOH:MTBE (40:60), were performed (Table

    2). Solubility of -carotene in MTBE was about 620 mg/L, somewhat lower than found in the

    literature [45], the difference may be due to that they have measured the solubility by UV/VIS

    spectrometry. The MeOH:MTBE solubility was determined at about 230 mg/L. To achieve the best

    possible solubility in the solvent, the -carotene was first dissolved in MTBE, filtered and methanol

    was then added to a given amount. The solubility problem was the reason not to choose the same

    proportion for MeOH:MTBE in the solvent as in the mobile phase. With methanol in a higher

    percentage -carotene immediately precipitated in the solvent. Methanol and the extraction solvent

    ethanol exhibit according to literature solubility at 10 mg/L and 20 mg/L respectively [45], this was

    confirmed by recognizing -carotene perceived almost insoluble in methanol and slightly better in

    ethanol.

    Table 2 Solubility for -carotene in different solvents.

    Solvent Experimental

    mg/L

    Literature

    mg/L

    MTBE 650 1000

    MeOH:MTBE 230 -

    MeOH almost none 10

    EtOH - 20

    3.3 Stability of -carotene

    3.3.1 BHT-studies

    The small BHT study performed during a period of this project show no significant difference between

    the uses or not of BHT to the -carotene solution. To keep it on the safe side it may be a good decision

    to use some antioxidant for long-term savings of -carotene solutions due to its ability to react. The

    same study establish the importance of store the -carotene solution in the freezer between the

    measurements, there is an significant difference between doing so or keeping it in room temperature.

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    3.3.2 Heat stability

    The heat stability test contradicted -carotenes instability while boiling. The test exhibits no

    considerable alteration of -carotene concentration during the two hour boiling session (CV= 12.42%)

    (Figure 10).

    Figure 10 Shift in -carotene concentration during heat stability test.

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    3.4 Increasing the concentration of -carotene

    Having isolated the -carotene from the carrot extract made by pressurized fluid extraction further

    increase the -carotene concentration of the solution is desirable. The original extract concentration of

    -carotene was determined to between 80-113 mg/L, in weight unit between 5.3-7.5 mg/100g. Which

    seem reasonable because according to literature the -carotene content after carrot extraction are

    reported to be in a range between 4.8-18.0 mg/100g [33, 42, 46, 47, 48, 49]. For a more commercial

    use the original concentration will neither be profitable nor environmentally friendly, with too much

    solvent consumption in relation to amount -carotene. Usually the largest possible quantity is solved

    in a small amount of solvent and the restriction is in the chromatographic system. The maximum is

    reached when the chromatographic system is overloaded. In this study this does not seem to be a

    feasible way to provide a commercial viable carotene solution due to the low solubility of -carotene

    in the selected solvent.

    3.4.1 Solid Phase Extraction

    For this approach it seems to work to increase concentration by solid phase extraction (SPE), four

    times higher -carotene concentration may be achieved, 288 mg/L from the original concentration at

    78 mg/L (Table 3).

    Table 3 -carotene (main sample) concentration and yield after solid phase extraction.

    mL through

    column

    Concentration

    main sample

    (mg/L)

    Concentration

    main sample

    per mL (mg/L)

    Yield (%) main sample from

    original concentration

    -carotene

    not.

    1 60.9 60.9 1,2

    3 236.5 78.8 2

    1 81.9 81.9 105.4 3,4

    1 79.3 79.3 102.2 3

    1 81.0 81.0 104.3 3

    5 287.6 57.5 74.1 3

    1 7 washing step.

    2 Original concentration uncertain (approx. 100.2 mg/L).

    3 Original concentration 77.7 mg/L.

    4 no further signal detected.

    The -carotene after SPE is solved in MTBE, one milliliter of MTBE for each five milliliter extract in

    ethanol, even if it is not the most environmentally friendly choice [50]. Ideally five times the original

    concentration should be achieved, e.g. given the solubility of -carotene in MTBE. This may not be

  • 26

    the case due to packing of the sample onto the column, however when one milliliter sample is set at

    the column all -carotene is eluted, yield 102-105% (Table 3), with good repeatability CV = 1.59 %.

    Due to lack of extract (from PFE) SPE test was also performed on a solution containing -

    carotene/MeOH. The purpose of this became to examine repeatability/reproducibility CV = 12.50 %

    of SPE for the main sample concentration per mL (50 mL not taken into account), concerning the yield

    this is not a suitable procedure (45-70 %), the overall yield is slightly better (67-89 %) (Table 4). The

    yield is as expected different from above due to methanol ability to solve such low concentration and

    thus more -carotene is lost in the washing step. 50 mL through column work unsatisfactory.

    Table 4 -carotene concentration and yield after solid phase extraction of -carotene/MeOH solution.

    mL through

    column

    Concentration

    main sample/

    per mL

    (mg/L)

    Total -carotene

    concentration

    through

    column(mg/L)

    Yield (%) main

    sample from

    original

    concentration -carotene

    Yield (%) main

    sample from total

    amount -carotene through column

    not.

    1 3.4/ - 3.6 70.2 74.3 a

    1 2.5/ - 4.3 63.7 89.5 a

    1 2.6/ - 4.1 54.4 84.5 1,a

    1 3.3/ - 4.2 58.5 73.7 b

    1 3.1/ - 4.4 54.2 77.8 b

    3 9.0/ 3.0 12.2 52.7 71.8 b

    5 16.1/ 3.2 16.8 56.6 68.8 b

    5 12.7/ 2.5 19.8 44.7 69.4 b

    5 13.0/ 2.7 16.9 45.7 59.3 b

    50 50.8/ 1.0 69.9 21.2 29.3 a

    50 63.6/ 1.3 112.3 22.4 39.3

    b

    Concentration -carotene/MeOH-lsning

    a 4.8 mg/L

    b 5.7 mg/L

    1 the solution flowed through the column three times.

    The optimum volume of methanol to wash the column was determined prior to the solid phase

    extraction. Two steps were decide to be the optimum, including more does not seem defensible, losing

    too much -carotene (Figure 11-13).

  • 27

    Figure 11 First washing step.

    Figure 12 Second washing step.

    Figure 13 Third washing step.

  • 28

    The main substances that disappear from the sample are sugars, it is a good idea to get rid of them

    before injection into the chromatographic system otherwise the sugar may clog the filter. Table 5

    includes the concentration -carotene detected in the sample prior to and after the main sample, this

    together with the total yield main sample.

    Table 5 Total -carotene concentration and yield of main sample through column.

    mL through

    column

    Total -carotene concentration through column (mg/L)

    Yield (%) main sample

    from total amount -carotene through column

    not.

    1 73.3 83.0 1

    3 240.9 98.2

    1 81.9 100.0

    1 82.4 96.3

    1 84.1 96.4

    5 294.3 97.7

    1 7 washing step.

  • 29

    3.5 Extraction of -carotene

    Initial comparison between the two methods Soxhlet and traditional boiling, reveals a small advantage

    in concentration after boiling, but counting the -carotene yield they exhibit an almost equal amount,

    about 17 mg from 100g carrot (Table 6). (According to the test results (not shown in the table) an

    overestimation of concentration towards what would be possible due to solubility in ethanol were

    achieved when the samples was not pooled.)

    Table 6 Comparison between extraction methods, time and content achieved.

    Refinement of the method eventuates in reflux boiling, due to the avoidance of solvent consumption

    by evaporation. A closer look reveals reflux boiling to be the most favorable, achieving the highest

    amount of -carotene in correlation with the lowest time used, 11.5g/100g carrot, extraction time

    between 5-10 minutes (Table 6, 7). 30 minutes were used for reflux, but the content does not improve

    after 5-10 minutes. When using the other two method 30-60 minutes results in a higher yield, but

    using about 10 minutes for each reflux, will give a higher total amount of -carotene during the same

    time. Incidentally the time used for reflux was well enough, no improvement during longer extraction

    time were seen for the smaller amount of carrot.

    Comparison between the amount -carotene in the extract achieved from the reflux boiling (11.5

    mg/100g) and the extract achieved from pressurized fluid extraction (5.3-7.6 mg/100g) (Table 6)

    resulted in the conclusion that a simple reflux boiling is well enough for extraction of -carotene when

    dealing with ethanol. The reflux boiling method is environmentally friendly according to the use of

    ethanol, even if it results in higher solvent consumption then pressurized fluid extraction. Due to the

    low accumulation of by-products recycle the ethanol hopefully will be possible. However, in this study

    the carrot preparation is more environmentally friendly without any requirement of desiccant, this

    according to the use of a juice extractor rather than a food processor.

    Extraction method Extraction time (min) -carotene content (mg/100g)

    Traditional boiling 230 17.0

    Reflux boiling 30 11.5

    Soxhlet 60 17.6

    Pressurized Fluid

    Extraction

    52 5.3-7.6

  • 30

    Figure 14 Carrot precipitate post extraction.

    Table 7 show a comparison between different amount of carrot used for the reflux extraction, the

    conclusion is to use 30 g carrot in 200 mL ethanol to get the maximum yield, larger amount does not

    improves in commensurate. The use of 50 g even exhibit a lower yield, likely owing to a larger

    amount of water, since larger amounts of carrot implies larger amounts of water (the carrot was not

    entirely dehydrated during extraction), which makes the solubility of -carotene in the solvent even

    poorer. Carrot post extraction is demonstrated in Figure 14.

    Table 7 Different carrot amount and optimum extraction time used during -carotene extraction in 200 mL ethanol.

    Amount carrot (g) Extraction time (min) Concentration (mg/L) -carotene (mg/100g)

    20 5 12.6 10.9

    20 5 12.6 11.2

    30 5 19.0 11.6

    30 5 18.5 11.1

    40 10 21.9 10.2

    50 30 18.1 6.9

    The concentration of the extract from the pressurized fluid extraction was higher than achieved in this

    study, but unlike the other the estimation here is done when all -carotene is dissolved in the ethanol.

    As mention before solubility of -carotene in ethanol is stated around 20 mg/L [45], which results in

    the conclusion that a concentration, for instance 80 mg/L of -carotene would not be possible in

    ethanol and must contain some precipitate. When dissolving the original solution containing the

  • 31

    precipitate further a homogenous portion is hopefully used, (this will possibly be a reason to the

    variable results from the concentration determination).

    Furthermore the solubility in ethanol (Table 2) seems to be a reason for the maximum amount of -

    carotene regardless to amount of carrot is between 18-22 mg/L (Table 7).

    Use of other solvents e.g. tetrahydrofuran (THF) may improve the extraction and certainly the

    solubility of -carotene [45]but to maintain this procedure in a reasonably environmentally friendly

    manner, this is not a good alternative [50]. The main disadvantage with THF ought to be its ability to

    form highly explosive peroxides when stored in air, this risk may however be reduced by inhibition of

    an antioxidant e.g. BHT. Nevertheless, as a food additive THF is not desired, since it in addition to

    possibly forming peroxides in general exhibit an unfavorable effect on the human body [39].

    The not environmentally friendly choices of solvents are in particular those with the advantage in

    terms of solubility of -carotene, in addition to THF and MTBE, benzene and halogenated solvents are

    favorable [45]. They are not either any attractive selection due to their overall impact on the

    environment and that benzene is suspected to cause cancer [39, 51]. The more environmentally and

    thereby often healthier (at least at low concentration) to human choices of solvents e.g. ethanol and

    propanol [50, 51], exhibit poor solubility [45] and are not of use in studies who striving for good

    solubility of hydrocarbons.

    In terms of money the best choice is methanol, considerably cheaper than other solvents. Both

    MTBE and THF will results in higher costs, while other e.g. ethanol, toluene, dichloromethane and

    heptane are somewhere in between [52]. According to these facts using methanol or ethanol would be

    a good choice when not working with e.g. hydrocarbons. In this study the use of methanol is not good

    enough and the use of ethanol in the extraction is not satisfactory in terms of profitability. To achieve

    the highest solubility of -carotene the choice of MTBE as the solvent was the most reasonable,

    although it is not the best.

  • 33

    4. Conclusions

    To accomplish the main aim of this study; purification and isolation of -carotene from the PFE

    extract, first a method for separation of -carotene was developed. The separation was achieved in

    only 2-3 minutes. Poor solubility in methanol resulted in the use of different solvent proportions in

    contrast to the mobile phase.

    SPE was used to increase the -carotene concentration, since the content of the PFE extract neither

    was profitable nor environmentally friendly. This resulted in a four times higher concentration than the

    original one, which is an enhancement although not as good as desired. This may be, as the solubility

    tests exhibit, due to the poor solubility of -carotene in the solvent, even despite MTBE was used.

    Extraction methods well comparable to the PFE method were developed, particularly reflux boiling

    who in 5-10 minutes gave a -carotene yield of 11.5 mg from 100 g carrot in contrast to PFE who in

    approximately the same time exhibit 5.3-7.6 mg/100 g. According to the solubility, the maximum

    amount carrot for reflux extraction was 30 g when 200 mL of ethanol was used. In the ethanol solvent

    the extracted -carotene reach a maximum concentration at 21 mg/L, which appears reasonable

    according to -carotenes solubility in ethanol. According to solubility of -carotene MTBE was used

    as the solvent in SPE and even better choices of solvents would be THF, nevertheless it is worse in

    terms of safety and to the environment, and also contributes to a high cost.

    Precautions when handling -carotene does not seem to improve the outcome of this study, as

    evidenced in heat stability and BHT studies. Though there may be good taking care since -carotene is

    easily reacting and to actually confirm the pointlessness in using BHT more long term studies should

    be performed.

    Further investigation should involve focusing on more specific task before any conclusions can be

    made or complete validated methods are achieved, this together with finding a more appropriate

    solvent for -carotene. If it seems appropriate, furthermore the objective might be to scale-up the

    process for industrial application. Another possibility is the usage of the purified compounds in

    research against diabetes and neurodegenerative diseases [1].

  • 35

    5. Acknowledgements

    You cant make a work entirely by your own, so I would like to thank:

    Jrgen Samuelsson, supervisor, for guidance in my work and assistance in the laboratory.

    Martin Ernmark, for help with the laboratory equipment.

    Torgny Fornstedt, supervisor and examiner, for advice in the writing of this paper.

    The people in Lund who give me PFE extract and supported me with extraction recipes.

    Everyone in my vicinity for showing interest and giving me support in my project inside and outside

    the laboratory, especially my boyfriend Niclas Dahlman.

  • 37

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