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

Karlstads universitet 651 88 Karlstad

Tfn 054-700 10 00 Fax 054-700 14 60
Information@kau.se www.kau.se
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
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.

9
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

10
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.

11
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.

12
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.

13
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.

14
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].

15
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.

16
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

17
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.

18
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.

19
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 =

21
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 0.30-0.60 %
standard (n = 5)
Repeatability of standards (n = 3) 0.79 %
Inter-day repeatability (n = 5) 2.50 %
LOD (mg/L) 0.22
LOQ (mg/L) 0.73

22
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 Literature

mg/L 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.

23
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.

24
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 Concentration Concentration Yield (%) main sample from not.

column main sample main sample original concentration
(mg/L) per mL (mg/L) β-carotene
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

25
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 Concentration Total β- Yield (%) main Yield (%) main not.
column main sample/ carotene sample from sample from total
per mL concentration original amount β-carotene
(mg/L) through concentration β- through column
column(mg/L) carotene
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).

26
Figure 11 First washing step.

Figure 12 Second washing step.

Figure 13 Third washing step.

27
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 Total β-carotene concentration Yield (%) main sample not.

column through column (mg/L) from total amount β-
carotene through column
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.

28
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.

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 5×2 5.3-7.6
Extraction

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.

29
 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

30
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.

31
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].

33
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.

35
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