Aqueous Size Exclusion Chromatography of Tobacco

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					Aqueous Size Exclusion Chromatography
          of Tobacco Extracts

Barbu A . Demian
Ph i I i p Morr i s USA
Research & Deve I opment
POB 26603, Richmond, VA, USA



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INTRODUCTION
~ 3 main varieties of smoking tobacco: Virginia (V)
                                       Kentucky (K)
                                       Oriental (0)

~ Water extractable components in the cured tobacco leaf :
        inorganic salts
        low MW organic acids
        alkaloids: nicotine, nor-nicotine, nicotyrine, myosmine
        polyphenols and glycosides
        carbohydrates : mono-, di-, and oligo-saccharides


~ Variability in the composition : variety, V vs. K vs . 0
                                    stalk position, crop year
                                     and place, curing practices
      s MM coz             etc .
~ Size Exclusion Chromatography - SEC [1]
    close relative of GPC - gel permeation chromatography
    for polymers
    gel filtration chromatography - GFC
    using soft gels - biochemistry
    "molecular sieveing"
    separation based on differences in the hydrodynamic
    radius of the molecules in solution



~ SEC of water extractables
    from tobacco for fingerprintig



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PROCEDURES

Extraction -> HPLC -> Data analysis


~ Extraction - water, 0 .1 M Na2HPO4 aq., 5% methanol/
                        water, 20% DMSO/water
                        1 hr. on wrist-action shaker
                        centrifugation - Z-spin filters 0 .45 ,c,am
                        cut-off


~ HPLC - isocratic, mobile phase = extraction solution
                    flow rate 0.4 to 1 .0 ml/min
                    run time 15' to 1 hr ., 30 min. typical
                    column temperature 40°C
                    RI detection

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Equipment: Waters 510 pump, WISP 712 injector
            ERMA ERC-7510 RI detector
  Data acquisition - Waters Maxima 820 or Nelson 2060
Columns:
                                     MW cut-off
PL Aquagel-OH 40                     > 100,000
Asahipak GS-220H                      3,000
Shodex OHpak KB-802                  4,000
              KB-803                  100,000
        CHITOpak KQ-802 .5                7,000
Jordi Gel DVB 500A GBR


~ Data analysis : "Lab-Caic" & PLS-Plus, Galactic Ind .
                  °Unscrambler", Camo
                  "Systat", SPSS Inc.
                  "Design-Ease", Stat-Ease, Inc .
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RESULTS ANS DISCUSSION

* Fingerprinting

   12 samples: V 1, V2, K 1 & K2
   Asahipak GS-220H / water
   Nelson 2060, 1 min. area slices
   from 10 to 24 .0 min .RT

Example chromatogram :
X Area Log hot F}t vs . Areal-Cur:!
.344053



                                      75




                                      5B



                                      25




ho1 Nt 6        5
     18    1B       184



V 1, 5g./ 150m1 water
Data analysis : Systat [2]
* Factor analysis : 4 factors - 97 .3% of variance explained


Plots of factor scores
factor(1) vs. factor(2) vs. factor(3)             factor(2) vs . factor(1)




                                         2




                                         1




                                                                         ~~




                                        -2                                   I
                                             -2                  0           1   2
                                                             FACTOR(1)



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Cluster Analysis
Single linkage, Nearest Neighbor, Euclidian distance

                                                     DISTANCES
                      n,npn                         5p0,n00
                       q
                             -_________________________

                      ti--- I                    I
                       +--                       ~
                      5---
                                                      t___________________________________
                      11---                       I
                          ~-
                      1=---I
                      LO___~______________________I I




                                ~-            I

                                 +-            I

                       L------' ~ ------------- I
                       1---- ~
                         +-
                      0----I I
                             ~-----




1,2,3: Virginia[1]               4,5,6 : Kentucky[ 1 ]
7,8,9 : Virginai[2]              10,11,12: Kentucky[2]




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CONCLUSION


Size Exclusion Chromatography profiles of tobacco
water extracts are fingerprints of the leaf variety




LITERATURE

[1] S. C. Chu ns, J. Chromato gr. A, 720. 151-66 (1996)

[2] L Wilkinson, MA. Hill, J.P. Welna, GK. Birkenbuel, SYSTAT for Windows,
    Version 5 Edition, Evanston, IL; SYSTAT, lNc. 1992




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