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

Information@kau.se 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

Abstract

With focus on purification and isolation, the fat soluble, highly conjugated hydrocarbon β-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 β-carotene. β-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 β-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 β-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 på rening och isolering har det fettlösliga, starkt konjugerade kolvätet β-karoten utforskats.

Ursprunget till studien är en del i ett forskningsprojekt vars syfte är att bättre ta vara på jordens

resurser, framförallt genom förädling av restprodukter ifrån industrin vilket inkludera bl a. β-karoten.

β-karoten uppvisar provitamin A aktivitet, är potentiellt sjukdomsförebyggande samt används som

färgämne inom olika delar av industrin, de aktuella användningsområden har resulterat i att β-karoten

är en eftertraktad förening. Källan till β-karoten för studien var först och främst morotsextrakt

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

genom vätskekromatografi (HPLC). För att utgöra ett lönsamt och miljövänligt alternativ bör extraktet

uppvisa en tillräckligt hög β-karotenkoncentration. Genom fastfasextraktion (SPE) erhölls fyra gånger

högre koncentration i förhållande till ursprungslösningen. Under studien utvecklades också

jämförande extraktionsmetoder, en metod baserad på återloppskokning uppvisade, genom mängden

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

påvisat att β-karoten vid hantering är instabil och lätt bryts ned på grund av att den har lätt att reagera,

faktum som tillbakavisa på flera punkter i den här studien så som β-karotens värmeinstabilitet och

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

långsiktiga studier för att eventuellt bekräfta det överflödiga i att använda BHT. Vidare forskning och

utvärdering av SPE-metoden och extraktionsmetoderna är också nödvändigt före en uppskalning av

processen för industriella tillämpningar.

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 β-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 β-carotene ............................................................................................................... 23

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

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

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

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

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

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

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

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

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

9

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.

10

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 π-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. β-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 β-carotene

β-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

11

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

investigated and correlate the carotenoids, particularly β-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 β-

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

will be continued.

Figure 3 Molecular structure of β-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 β-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 β-carotene and further scale-up the process.

The main aim of the study was to purify and concentrate β-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 β-carotenes stability.

12

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)

ΔtR 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.

13

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.

14

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.

15

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].

16

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.

17

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). β-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 β-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 µL (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 µm particles) Kromasil (Eka Chemicals AB, Bohus, Sweden)

interconnected with a filter PEEK end fitting 0.5 µm 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 β-carotene was achieved isocratically with

the C18 column kept under constant temperature (22.9°C set at 21°C) 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 β-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 β-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

18

instability of the β-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 2×100

mL ethanol for 2×30 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 60°C,

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 (2×1 mL) was used to

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

2.8 Solubility test

β-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.

19

2.9 BHT studies

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

the influence of BHT on β-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

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

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

temperature constant at 70°C. 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.

21

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 β-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 β-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 β-carotene is clearly identified.

3.1.2 Linearity and Precision

The developed analytical method showed linearity for β-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 β-carotene concentration, on to the following day the sample showed no differences CV =

22

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 β-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

23

3.2 Solubility of β-carotene

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

2). Solubility of β-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 β-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 β-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 β-carotene perceived almost insoluble in methanol and slightly better in

ethanol.

Table 2 Solubility for β-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 β-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 β-carotene solution. To keep it on the safe side it may be a good decision

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

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

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

24

3.3.2 Heat stability

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

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

(Figure 10).

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

25

3.4 Increasing the concentration of β-carotene

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

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

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

78 mg/L (Table 3).

Table 3 β-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

β-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 β-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 β-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 β-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 β-

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

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

mL through

column

Concentration

main sample/

per mL

(mg/L)

Total β-

carotene

concentration

through

column(mg/L)

Yield (%) main

sample from

original

concentration β-

carotene

Yield (%) main

sample from total

amount β-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 β-carotene/MeOH-lösning

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

together with the total yield main sample.

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

mL through

column

Total β-carotene concentration

through column (mg/L)

Yield (%) main sample

from total amount β-

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 β-carotene

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

in concentration after boiling, but counting the β-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 β-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 β-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 β-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 β-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) β-carotene content (mg/100g)

Traditional boiling 2×30 17.0

Reflux boiling 30 11.5

Soxhlet 60 17.6

Pressurized Fluid

Extraction

5×2 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 β-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 β-carotene extraction in 200 mL

ethanol.

Amount carrot (g) Extraction time (min) Concentration (mg/L) β-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 β-carotene is dissolved in the ethanol.

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

the conclusion that a concentration, for instance 80 mg/L of β-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 β-

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 β-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 β-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 β-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 β-carotene from the PFE

extract, first a method for separation of β-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 β-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 β-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 β-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 β-carotene reach a maximum concentration at 21 mg/L, which appears reasonable

according to β-carotenes solubility in ethanol. According to solubility of β-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 β-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 β-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 β-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 can’t make a work entirely by your own, so I would like to thank:

Jörgen 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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