: Essentialsin "

950¢$5

Poster Presentation

FASEB/ASBMB Symposium - April, 1991Poster # 8055

Non-Aqueous Reverse Phase Purificationof Carotenes on a Small Particle

Preparative Packing

K. O'Connor & G. VellaMillipore Corporation, Waters Chromatography Division _

34 Maple Street, Milford, MA. 01757 U.S.A.

-'f

Woters Chromatography Division Waters 'v/Waters. The absolute essential MilliporeCorporationfor bioresearch. 34 Maple St. Div,s,ono[ MILLIPORE

Milford, MA 01757(508) 478-2000

7

9., - Introduction

Carotenoids are a class of naturally occurring hydrocarbons consisting mostly of

C40 molecules varying in the degree of conjugated double bonds. They are ubiquitous

in nature and can be found in fungi, bacteria, photosynthetic and non-photosynthetic

plant tissue such as algae and seaweed. Some birds, fish, amphibians and reptiles owe

their vivid colors to carotenoids. Carotenes and xanthophyUs are subclasses of

carotenoids; the xanthophylls are oxidized forms of the carotenes and both can be dark

red to yellow in color. Figure 1 shows a few of the carotene structures and their

absorbance maxima.

Due to the conjugated nature of these molecules considerable interest in the

carotenoids has initiated research endeavors in elucidating the role they play in

photosynthetic processes, vitamin A activity in vision and more recently beta-carotene's

role as an anticarcinogenic agent 1. Carotenes are also used as food additives,

antioxidants or are available as food supplements. Hence the need for large amounts of

pure carotenoids is of paramount importance.

Both analytical and preparative methods for the separation of the classes of

carotenoids have been reported in the literature2-7. These methods have utilized thin

layer chromatography, as well as normal phase and reverse phase column

chromatography. Each method has certain advantages and disadvantages and the

conditions of elution depends on the sample matrix and the particular carotenoids to be

separated or isolated.

Described here is a non-aqueous reverse phase method which separates

configurational carotene isomers extracted from a carrot homogenate, and also permits

resolution between cis and trans isomers of beta-carotene. In addition this methodology

is amenable to scale-up so that larger quantities of purified compounds can be isolated.

Methods .v

Sample Preparation

Raw Carrots

Blend w/MeOH& Filter

/Carrot Pulp MeOH ( Discard )

Wash with THFUntil Colorless

Pulp ( Discard ) THF Concentrate

Evaporate to Dryness

Crude Carotenes

Dissolve in 25%Toluene75% Mobile Phase

HPLC Analysis

i

li •

Optimization of Analytical Separation:

The separation of the carotenoids was first optimized on a high resolution

analytical column (3.9 x 300mm) packed with 4grn Nova-Pak C18 material

(Figure 2 A,B). Identification of the various carotenoids in the crude mixture

was confirmed by comparing relative retention times to known standards and by

their characteristic UV/Vis absorbance spectra which were obtained by using an

on-line Waters 994 photodiode array detector. Spectra were collected between

200nm and 600nm and were critical in the identification of several of these

carotenoids.

Scale Up:

The optimized conditions on the analytical column were transferred to a scaling

column ( 8 x 100mm, cartridge column ) packed with a small particle preparative

packing, 6gm Prep Nova-Pak HR C18 (Figure 2C). A loading study was carried

out to determine the maximum amount of sample this column could tolerate

under these conditions without compromising resolution of the components of

interest. Once established, the flow rate and maximum load on the scaling

column can now be used to determine the conditions to be employed for any

larger volume column packed with the same particle size and of the same length.

The sample load and flow rate are increased proportionally to the cross-section of

the packed bed 8 as shown in Equations 1 and 2.

!

Scaling Equations '

Mass (prep) / Mass (scaling) - r2 (prep) / r2 (scaling) ( 1 )

Flow (prep) / Flow (scaling) = r2 (prep) / r2 (scaling) ( 2 )

r = column radius

If both column diameter and column length are changing, the flow rate and

sample load can be changed proportionally to column volume, as shown in

Equations 3 and 4 below:

Flow (prep) = Volume (prep) = Length (prep) * r2 (prep) ( 3 )

Flow (scaling) Volume (scaling) Length (scaling) • r2 (scaling)

Mass (prep) = Length (prep) • r2 (prep) ( 4 )

Mass (scaling) Length (scaling) ° r2 (scaling)

This results in identical chromatographic performance ( retention times, peak

widths and resolution ) at the preparative scale as that of the scaling column. The

flow rate and the maximum sample load on the scaling column were found to be

1.0ml/min ( 4.0cm/min linear velocity ) and 1.2mg ( 150gl ) respectively.

Employing equations 1-4 the flow rates and the loads were calculated for three

column cartridge configurations, packed with 6gm Prep Nova-Pak HR C18

material, which were used in a stackable cartridge holder ( Table 1 ).

Chromatography is shown in Figure 3 A,B,C. Fractions were collected during

the preparative separation using 25 x 300 cartridge configuration ( Fig 3A ).

i

t Results and Discussion

Aliquots ( 50-200gl ) of the fractions collected during the preparative separation

were analyzed using the high resolution 41.tNova-Pak column. Samples were injected in the

form they were collected i.e. without evaporation or alteration ( Figures 4-8 ). Fraction 8

was identified as phytoene based on its strong absorbance a 286nm3. This spectrum is

shown in Figure 4. Fractions 1 and 2 have been tentatively identified as cis-zeta-carotene

and trans-zeta-carotene based on their UV/vis spectral maxima and retention relative to the

other acyclic carotenes 5 ( Figures 5 and 6. ).

Fraction 3 ( Figure 7 ) was identified as alpha-carotene by its retention and spectral

characteristics which were identical to those of a standard. These three fractions together

with phytoene represent pure components. Fractions 4,5,6 and 7 contained approximately

80%, 90%, 65% and 30% trans-beta-carotene as the spectral characteristics were very

similar to those of a trans-beta-carotene standard. These fractions also exhibited

absorptions between 310 and 390nm which had three maxima at 33 lnm, 347nm and 366nm

in addition to absorptions attributable to cis-beta-carotene. A comparison of the three

maxima matched those of phytofluene3,5.

The amount of phytofluene and cis-beta-carotene appears to vary from fractions 4

to 7 suggesting that more than one isomer of each may be present. Several isomers are

known to exist for both phytofluene and cis-beta-carotene5,6. Therefore it is suggested that

fraction 4 contained predominantly trans-beta-carotene with a small amount of phytofluene

and cis-beta-carotene. Fraction 5 appears to be most enriched with trans-beta-carotene

( >90% ) with a small amount of phytofluene. ( Figure 8 ) Fractions 6 and 7 contained

decreasing amounts of trans-beta-carotene and what may be other isomers of cis-beta-

carotene and phytofluene. Yet the fact that similar spectra are observed from

noncontiguous fractions suggest that these other isomers are being resolved from each other

however, they coelute with other carotenes found in this mixture.

J

Conclusion

The difficulty in performing a separation of this type is that all these

compounds are closely related and cannot be separated using one simple

chromatographic method. The separation must be developed to isolate

components of interest. An analytical method for the separation and

identification of carotenes in carrot homogenate has been presented. This

methodology can be used to scale to larger preparative cartridges and can be used

to analyze fractions collected from the preparative separation. The large scale

purification of the carotenoids using a @m preparative reversed phase packing

material permitted the isolation of five different compounds as pure components.

Nine different carotenes were tentatively identified by their spectral

characteristics and relative retention times. Positive identification of these would

require the aid of additional structural analysis such as NMR or mass

spectroscopy 5. Alternative mobile or stationary phases may afford sufficiently

different selectivities, thereby resolving compounds which coelute or to separate

components not addressed here or from other sources.

References:

1. Burton, G.W. and Ingold, K.U., beta-Carotene: An unusual type of lipidantioxidant. Science 224, 569-575, (1984)

2. Neils, H.J. and De Leenheer, A.P., Isocratic nonaqueous reverse phase liquidchromatography of carotenoids. Analytical Chemistry. 55, 270-275, (1983)

3. Tan, B. and Andersson, A., Analytical and preparative chromatography oftomato paste carotenoids. J. Food Science, 53(3), 954-959, (1988)

4. Bushway, R.J., Separation of carotenoids in fruits and vegetables by highperformance liquid chromatography. J. Liquid Chrom., 8(81, 1527-1547, (1985)

5. Davies, B.H., Carotenoids In " Chemistry and Biochemistry of PlantPigments," T.W. Edwin (Ed.) Vol. 2, pg. 38 Academic Press, Orlando FL.(1976) and references therein

6. Quackenbush F.W., Reverse phase HPLC separation of cis- and trans-carotenoids and its application to beta-carotene in food materials. J LiquidChrom., 10(4), 643-653, (1987)

7. Scalia, S. and Francis, G.W., Preparative scale reversed-phase HPLC methodfor simultaneous separation of carotenoids and carotenoid esters.Chromatographia, 28(3,4), 129-132, ( 1989 )

8. Neue, U. Private Communication (1990)

Table 1.

Number of Cartridges. Column Volume Flow Rate Sample Load Run Time

1 x ( 8 xl00mm )* 5.0mls 1.0ml/min 1.2mgs (150_tl) 35rain

1 x ( 25 xl00mm ) 50.0mls 10.0ml/min 12.0rags (15001al) 35min

2 x ( 25 xl00mm ) 100.0mls 20.0ml/min 24.0mgs (30001al) 35min

3 x ( 25 xlOOmm ) 150.Omls 30.Oml/min 36.0mgs (45001.tl) 35min

* Scaling cartridge column ( 8 x 100mm )

v

q _Q

Figure 2" Scale-up of Crude Carrot Extract from ,t_Analytical to Preparative Particle Sizes ,.

Column: Walers Nova-Pak "rMC! 8, 4p.m, 3.9mm x 300mm ....Mobile Phase: CH3CN/CH3OH/IHF 58/35/7

Flow Rale: ].Oml/min alpha-carotenoTemperalure: 30.0 C cis-zela-carolene / ./_ lrans-bela<orotene

d .25

AU 0.20 - 400nm 2A

0.15 _ ela-corolene0._0

0.05 _,..,__ -0.00 i I I r .................. 9-

AU

i 0.2 L

0 . 0 , , i f , " ' ' ' I ,--r-_ • T--r-'-t--', , I , -1--.r- , , _ _ , , I ' T---r--1--nr-_,'-*- , , I T'-r_-r--,_-'r -rl--_r--l , -r-T-r'-r--r_

0 5 :1.0 '15 20 25

Column: 8mm x l OOmm RadiaI-Pak"rMCarlridge packed with Prep Nova-PakrM C18,6pmMobile Phase: CH3CN/CH3OH/IHF 58/35/7Flow Rate: 1.Oml/minTemperature: 30.0 C

AU

O. 4 - 40Onto A 2C0.2

J_ ..0.0 i i T --T-- !

0 4OMinules

Figure 4: Analysis of Fraction 8 Containing Phytoene.. ".tl

t

Column: Waters Nova-PakTM C18, 4t_m,3.9mm x 300mm,._J Mobile Phase: CH3CN/CH3OH/THF 58/35/7

FlowRate: 1.0ml/min0.035 Temperature: 30.0 C

Detection:UV @286nm0.030

0.025

0.020,

0.015 -

0.010-

0.005 -

0.000

0 5 I0 15 20 25 30 35}_nutes

Fraction8 (46.9-49.2 UV/Vis @26.0min)

AU

0.8 A0.6

0.4

0.2

0.0

2oo 2_os6o s._o46o 4._o560 5._o60orim

uv/vis spectrumobtained at peakmaximaduring analytical separation

Figure 5" Analysis of Fraction 1 Containing cis-Zeta-' Carotene.

1. _ ,.AU Column: Waters Nova-PakTM C18, 41_m,3.9mm x 300mmo,

0.14 Mobile Phase: CH3CN/CH3OH/THF 58/35/7Flow Rate: 1.0ml/min

" Temperature: 30.0 C0.12 - Detection"UV/Vis @400nm

0.10 -

0.08-

0.06 -

0.04 -

0.02

0.00=

I I I I I

0 5 I 0 15 20 25_nutes

rim

Fraction1 ( 32.4-33.8 UV/Vis @18.9min )

AU

0.10.

0.05 -

0.0

200 250 ' ' ' '300 350 460 4so 500 5._06oonm

UV/Vis spectrumobtained at peakmaxima during analytical separation

Figure 6" Analysis of Fraction 2 Containing trans-Zeta- ,

Carotene. I'Column: Waters Nova-PakTM C18, 4_m, 3.9mm x 300ram "

0.,035- Mobile Phase: CH3CN/CH3OH/THF 58/35/7FlowRate: 1.0ml/min iTemperature: 30.0 C

0.C33 - Detection:UV/Vis @400nm

0.025 -

0.02O -.

0.015 -

0.010 -

0.005 -

0.000 -i

I i i i I

0 5 10 15 20 25Minutes

Fraction2 ( 34.6-35.4 UV/Vis @19.5rain )

AU 0.8

0.6.

0.4-

0.2.

0.0.

I I I I i I i

200 250 300 350 400 450 500 550 600nrn

uv/vis spectrumobtained at peakmaximaduring analytical separation

' . , Figure 7: Analysis of Fraction 3 Containing Alpha-t, Carotene.

o

, Column: Waters Nova-PakTM C18, 41.zm,3.9ram x 300ram' AU Mobile Phase: CH3CN/CH3OH/THF 58/35/7

0.060 .. Flow Rate: 1.0ml/minTemperature: 30.0 CDetection" UV/Vis @ 450nm

0.050 -

0.040 -

0.030 -

0.020 -

0.010 -

0.000 -

i i i " i I I I

0 5 10 15 20 25 25 25Minutes

Fraction 3 ( 36.9-38.4 UV/Vis @ 21.3min )

AU

0.1

0.05

200 250 300 350 400 450 500 600 600

rimUV/Vis spectrum obtained at peakmaxima during analytical separation

I Carotene. :

Column: Waters Nova-PakTM C18, 4_.m, 3.9mm x 300ram '0.060- Mobile Phase: CH3CN/CH3OH/THF 58/35/7

Flow Rate: 1.0ml/min' Temperature: 30.0 C

0.050- Detection: UV/Vis @ 450nm

0.040 -

0.030-

0.020-

0.010-

ClS0.000 _

dk --_._

I I I i I I I

0 5 10 15 20 25 30 35Minutes

Fraction.5 ( 40.9-41.7, UV/Vis 22.7min )

AU0.1

0.1

I ! I

200 250 300 350 400 450 500 550 600

rimUV/Vis spectrum obtained at peakmaxima during analytical separation

|, Figure 1Structures and absorption maxima ( nm )* of carotenes 8.

Beta- Carotene 425 450 477

Alpha-Carotene 420 442 472

Gamma-Carotene 437 462 492

Delta-Carotene 428 458 490

Zeta-Carotene 380 400 425

Lycopene 448 473 504

* in hexane

Figure 3" Preparalive Separalion of Crude CarrotExtract using Waters PrepPak® Cartridge Columns

Column: Waters Nova-PakrM C18, 6p.m25mm x 100, 200 and 300mm CartridgesMobile Phase: CH3CN/CH3OH/THF 58/35/7Sample: Crude Carrot ExtractTemperature: 25.0 CPump: Waters 600E Multisolvent Delivery SystemDeteclion: UV @ 400rimInjeclor: Waters U6K wilh a l Oral loop

AU _ A1.2 _ 25mm x 300ram @ 30ml/rnin, 4.5ml loaded_,.0

o.a Z F,°3I_ 3A,,°,

o4 _ A'?°U \/. \ _,.8o 2 _ __ ,/-f',F_--_,I_ IT---,-I---.]O. 0 i u i ....I 1

Aus.2 _ 25mm x 200mm @20ml/min, 3.0ml Ioa1.0 _o.e _ 3B0.6 _0.4

0.2 - _L-,. _ __ _

0.0 u, 1 I _ I-- .................

Aus.o - 25mm x lOOmm@ 1Oml/min, 1.Sml IoaO.e _ 3C0.6

0.,I _

0.2 __ ,0 0 '

0 10 20 30 40 50 " " ',

Minutesfo