Name: Locker Number:  

Experiment Title: Isolation of Lycopene and B-Carotene  Date: 0​ 1/28/2020 

 
Objectives: 
● To  extract  lycopene  and  b-carotene  from tomato paste via solid-liquid extraction and 
column chromatography. 
● To  characterize  the  extracted  samples  with  qualitative  tests,  thin-layer 
chromatography, and IR spectroscopy. 
 
Introduction: 
Lycopene,  a  bright  red  pigment  found  in  tomatoes  and  other  red  fruits  and 
vegetables,  is  a  C​40-​carotenoid  composed  of  eight  isoprene  units  (1).  On  the  other  hand, 
b-carotene,  an  isomer  of  lycopene,  is  the  yellow pigment found in carrots. The double bonds 
at  C​1​-C​2  and  C’​1​-C’​2 are replaced by bonds extending from C​1 to C​6 and from C’​1 to C’​6 forming 
rings  and  is  also  a  constituent  of  the  tomato  (2).  This  experiment  aims  to  extract lyc opene 
and  b-carotene  from  tomato  paste  via  solid-liquid  extraction  and  column  chromatography. 
Column  chromatography  is  a  chromatography  technique  used  to  separate  a  mixture  of 
chemical  substances  into  its  individual  compounds.  It  is  widely  used  for  the 
purification/separation of chemical compound mixtures (3). 
 
Solid-liquid  will  first  be  done  on  the  tomato  paste  using  acetone.  This  step  removes 
most  of  the  water  from  the  mixture.  After  the  aqueous solution has been decanted, multiple 
5-mL  portions  of  dichloromethane  will  be  used  to  extract  the  carotenoids  from  the  paste. 
The  dichloromethane-carotenoid  solution  will  be  dried  over  anhydrous  sodium  sulfate  to 
remove  any  remaining  traces  of  water.  Afterward,  it  will  be  filtered  into  a  small  flask.  TLC 
analysis  will  be  used  on  a  small  amount  of  the  crude  sample  to  select  the  hexane-ethyl 
acetate  solvent system that yields the best separation for the column chromatography. Once 
an  appropriate  solvent  system  has  been  selected,  a  column  chromatography  set-up  will  be 
used  to  purify  the  crude  sample.  The  collected  bands,  which  are  stored  in  separate  flasks, 
will  be  allowed  to  evaporate  to  dryness  under  a  fume  hood.  Once  dry,  the  purified  samples 
will  then  be  characterized  by  qualitative  tests,  thin-layer  chromatography,  and  IR 
spectroscopy (4).  
 
Reagents and Products:  
Reagent  Physical  and  Chemical  Chemical  Hazard and Safety 
Properties  Precautions 

(5) ● Clear, colorless liquid with ● Extremely flammable

Hexane a gasoline smell.
C​6​H​14 ● Insoluble in water.
● BP: 62-69 °C
● MP: -95 °C
● MW: 86.18 g/mol
● Density: 0.678 g/cm​3
liquid and vapor. May
cause flash fire.
● Vapors may cause
drowsiness.
● It causes eye, skin,
digestive, and respiratory
tract irritation.
● May be corrosive to
metals.
 ● Wear complete PPE.
● Do not breathe
dust/fumes/gas/mist/
vapors/spray.
● Wash skin after handling.

(5) ● Colorless liquid with a ● Extremely flammable

Propanone pungent, irritating odor. liquid and vapor.
Acetone ● Solubility: Miscible. ● Vapor may cause flash fire.
(CH​3​)​2​CO ● BP: 56.05 °C ● Causes eye, skin, and
● MP: -94.7 °C respiratory tract irritation.
● MW: 56.08 g/mol ● Harmful if inhaled. Vapors
● Density: 0.78 g/cm​3 may Cause drowsiness or
dizziness.
● Use with adequate
ventilation.
● Wear complete PPE.
● Do not breathe
dust/fumes/gas/mist/
vapors/spray.
● Wash skin after handling.

[cite ● White, crystalline solid. ● May cause eye, skin, and

Sodium sulfate ● Soluble in water. respiratory tract irritation.
Na​2​SO​4 ● BP: 1429 °C ● Hygroscopic.
● MP: 884 °C ● Use with adequate
● MW: 142.04 g/mol ventilation.
● Density: 2.67 g/cm​3 ● Wear complete PPE.
● Do not breathe
dust/fumes/gas/mist/
vapors/spray.
● Wash skin after handling.

(5) ● Colorless liquid, sweet ● Category 2 Eye Irritant

Ethyl acetate odor ● Category 2 Flammable
C​4​H​8​O​2 ● Slightly soluble in water. Liquid
● BP = 75-78 °C ● Highly flammable liquid
● MP = -83.5 °C and vapor
● MW = 88.11 g/mol ● Causes eye irritation; may
● Density = 0.902 g/mL cause drowsiness,
dizziness, dry skin,
irritation, cracking
● Keep away from any heat
source or ignition source.
● Wear complete PPE.
● Do not breathe
dust/fumes/gas/mist/
vapors/spray
● Wash skin after handling.

(5) ● Colorless liquid, ● Harmful if swallowed.

Methylene chloride chloroform-like odor
Dichloromethane ● Slightly soluble in water.
CH​2​Cl​2 ● BP = 40 °C
● MP = -97 °C
● MW = 84.93 g/mol
● Density = 1.33 g/mL
● Causes eye, skin, and
respiratory tract irritation
and possible eye burns.
● May be harmful if inhaled.
● Stable at room temperature
in closed containers under
normal conditions.
● May form explosive
mixtures in atmospheres
with high oxygen content.
● Avoid excess heat.
● Wear complete PPE.
● Do not breathe
dust/fumes/gas/mist/
vapors/spray
● Wash skin after handling.

(5) ● Colorless-white, odorless, ● Causes repiratory tract

Silicon dioxide solid granules. irritation.
Silica gel ● Insoluble in water. ● May cause skin, eye, and
SiO​2 ● BP = 2230 °C digestive tract irritation.
● MP = 1704.4 °C ● Do not ingest or inhale.
● MW = 60.08 g/mol ● Wear complete PPE.
● Density = 2.1 g/mL ● Do not breathe
dust/fumes/gas/mist/
vapors/spray
● Wash skin after handling.

(5) ● Deep red ● Slightly hazardous in case

Lycopene crystalline/powdered solid.
C​40​H​56 ● Insoluble in cold water.
● BP = 660.9 °C
● MP = 177 °C
● MW = 536.89 g/mol
● Density = 0.889 g/mL
of skin or eye contact,
ingestion, or inhalation.
● May be combustible at
high temperatures.
● Slightly flammable in the
presence of heat.
● Wear complete PPE.
● Do not breathe
dust/fumes/gas/mist/
vapors/spray
● Wash skin after handling.

(5) ● Dark orange ● Slightly hazardous in case

β-Carotene crystalline/powdered solid.
C​40​H​56 ● Insoluble in cold water.
● BP = 633 °C
● MP = 178 °C
● MW = 536.89 g/mol
● Density = 1.00 g/mL
of skin or eye contact,
ingestion, or inhalation.
● May be combustible at
high temperatures.
● Slightly flammable in the
presence of heat.
● Wear complete PPE.
● Do not breathe
dust/fumes/gas/mist/
 vapors/spray
● Wash skin after handling.

(5) ● Dark red paste with tomato ● Not considered hazardous.

Tomato Paste odor.
● Slightly soluble in water.
 
Reaction Mechanism: 
 
 
Procedures: 
Dehydration and Extraction of Tomato Paste: 
Test  tube  +  10g  tomato  paste  +7mL  acetone  (stir)  →  decant  or  filter  through  a  small 
Buchner  funnel  →  remove  from  tube  and  squeeze  as  much  liquid  as  possible  in  the 
funnel → discard yellow filtrate → test tube +solid residue +5 mL DCM → shake and filter 
(repeat  2-3x)  →  test  tube  +  filtrates  +  Na​2​SO​4  →  filter  into  small flask → reserve a small 
amount for TLC analysis → weigh crude material 
 
TLC analysis for solvent selection 
Perform  4  trials  of  thin-layer  chromatography  using  the  reserved  crude  samples  and 
varying  concentrations  of  hexane  and  ethyl  acetate  (0%,  1%,  3%,  and  5%  etOAc  in 
hexane).  →  prepare  a  500  mL  hexane:etOAc  solution  based  on  the  ratio  that  yields  the 
best separation. 
 
Preparing the Column Chromatography Set-up 
  
Open  column  →  loosely  plug  the  base  with  a  cotton  ball  →  fill the column beyond the curve 
of  the  base  with  sand  →  pour  5-10  mL  eluting  solution  →  250-mL  beaker  +30g  silica 
+hexane  2x  volume  of  silica  →  pour  silica  into  column  →  tap  the  side  of  the  column  to 
dislodge  air  bubbles  →  drain  excess  solvent  until  it  almost  reaches  the  top  of  the  settles 
silica  gel  layer  →  add  1cm  layer  of  sand  on  top  of  silica  →  Dried  crude  extract  +  1-2  mL 
eluting  solvent  →  uniformly  introduce  the  extract  into  the  column  with  a  dropper  or  pipette 
→  drain  colored  extract  until  it  almost  reaches  the  top  of  the  sand  →  add  a few mL hexane 
to the top of the column → drain until it almost touches the top of the silica layer 
 
Column Chromatography 
Continuously  elute  hexane-etOAc  solution  into  the  column  → note the color and order of 
any  bands  that  elute  →  as  bands  elute  from  the  column,  collect  in  separate  tared 
beakers → allow collected solutions to evaporate to dryness under a fume hood 
 
Characterization 
Spot  the  dry  separated  solution  into a TLC plate and elute with the hexane-etOAc solvent 
system. → combine purified products and conduct infrared spectroscopy 
 
Possible Hazards and Accidents: 
● Broadening  of  bands  during  column  chroma  →  only  add  additional solvent when the 
meniscus  of  the  sample  solution  is  close  to  the  surface  of  the  adsorbent.  This 
prevents dilution of the solution. 
● Formation  of  channels  in  the  column  →  always  make  sure  that the eluting solvent is 
kept above the top of the packed column  
● Inhalation  of  DCM  →  wear  masks  and  as  much  as  possible,  work  under  the  fume 
hood when using DCM. 
 
References: 
(1) May, M. E. What's Lycopene? https://www.poison.org/articles/lycopene-171
(accessed Jan 27, 2020).
(2) Olsen, N. Benefits of Beta Carotene and How to Get It.
https://www.healthline.com/health/beta-carotene-benefits​ (accessed Jan 27, 2020).
(3) Clark, J. Column Chromatography. ​https://www.chemguide.co.uk/analysis/
chromatography/column.html​ (accessed Jan 27, 2020). 
(4) Nijhawan, K.; Holt, P. PDF.
(5) Fisher Scientific. SDS. ​https://www.fishersci.com/us/en/catalog/search/sdshome.html
(accessed Jan 27, 2020).