Coupling multivariate statistical tools with analytical techniques for the study of copals in...

Team of Engineering applied to the Restoration of Ecological Cultural & Patrimony

Faculty of Sciences, University of Avignon, 33 rue Louis Pasteur 84000 Avignon

Paola [email protected]

Coupling multivariate statistical tools with analytical techniques for the study of copals in cultural heritage

Intensive School in Conservation Science – Biomaterials in cultural heritage

University of Avignon

26-28 Juin 2012

Thesis director : Pr. Cathy Vieillescazes

Co-director : Dr Carole Mathe

Bourse Doctorale CONACYT

Aim Study of terpene derivatives of exudates from Mexican tree

known as copals and belonging to Bursera spp. (Burseraceae family).

Analysis of:

Contemporary samples, botanically certified and

Archaeological Aztec samples of resinous materials



26-28 Juin 2012

Origin

Definition

Botanic : genus Bursera spp.

Over 80 species in Mexico

Sampling of resins of 7 botanically certified species

Chemical composition

Complex chemical mixture : its composition changes according with botanic and geographic criteria.

H

HH

23 24

25 26

27

28

30 29

H

HH

23 24

25 26

27

28

29

30

A B

CD

E

H

H

23 24

25 26

27

29

30

28

H

oleanan

ursan

lupan

Definition of Copal



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B. stenophyllaB. bipinnata B. penicillataB. simarubaB. grandifoliaB. excelsaB. laxiflora

Geographical Distribution



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Collection of the resin

B. bipinnata tree



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Aztecs founded Mexico-Tenochtitlan city on XIVth century.

Templo mayor

Hernán Cortés

Coyolxauhqui

Archeological Context



a

bd

c

a) Templo Mayor Pyramid

b) Cuauhxicalco

c) House of the “Eagle warriors”

d) Area explored by

archaeological excavations

Archeological Context

Reconstruction of Mexico-Tenochtitlán according to Antonio Serrato Combé



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Presentation of archeological samples

Thirteen Aztec offerings

6 samples, of an average mass of 5 mg

Suitable analytical strategy



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Modern sampleModern sample Archeological SamplesArcheological Samples

References for modeling behavior of the material

References for modeling behavior of the material

FTIR AnalysisFTIR Analysis HPLC AnalysisHPLC Analysis

PCA-LDA couplingPCA-LDA coupling

Archeological object of

study

Archeological object of

study

Archeological Site

Archeological Site

Characterization of resinous materials

Characterization of resinous materials

Interpretation of the results

Interpretation of the results

Analytical Stategy



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These results indicate that each botanical species has a different

FTIR spectrum.

.

FTIR (Fourier Inverse Infrared) analysis

4000 3500 3000 2500 2000 1500 10000

100

200

300

400

500

B. stenophylla

B. bipinnata

B. penicillata

B. excelsa

B. laxiflora

B. grandifolia

Tra

nsm

itance

(A

.U.)

Wavenumber (cm -1)

Molecular profile of resins =

"identity card"

Band position cm-1

Interpretation

a 3425 Tension ν O-H broad band

b 2945Tension ν C-H from alkans CH3 and alkens CH2

c 1710-1720

Stretching ν C=O from carboxylic acid

d 1638 Tension ν C=C

e 1454 Symmetric deformation δ CH2

f 1380 Deformation of deflection δ CH3

g 1242 Tension ν C-O-C

h 1037 Tension ν C-O symmetrical from alcohol

i 883

j 687

k 584



Multivariate Statistical Tools: PCA analysis

23002250220021502100

720

700

680

660

640

620

First component

Seco

nd c

ompo

nent

2 first components of FTIR data of contemporary certfied resins

Species are shown with different symbols: ●B. laxiflora, ♦B. excelsa, ■B. stenophylla, ▲B. bipinnata, ►B. grandifloia, ▼B. penicillata.



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LDA

Allows the construction of model able to foretell the property of a sample to a category previously defined.

LDA AnalysisPut into Group

bipinnata

excelsa

grandifolia

laxiflora penicillata stenophylla

bipinnata 8 0 0 0 0 0

excelsa 0 4 0 0 0 0

grandifolia 0 0 6 0 0 0

laxiflora 0 0 0 10 0 0

penicillata 0 0 0 0 6 0

stenophylla 2 0 0 0 0 8Total N 8 4 6 10 6 8N correct 6 4 6 10 6 8Proportion 0.75 1 1 1 1 1

N = 42 N Correct = 40 Correct Proportion = 0,952

Confusion matrix & Fitting Matrix (identical) of the LDA classification model: rows represent the true class, and the columns the assigned class



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Aplication to Archeological Samples

23002250220021502100

720

700

680

660

640

620

First component

Seco

nd c

om

ponent

2 first components of FTIR data of contemporary certfied resins and archeological Aztec samples

Aztec archeological samples

are shown as follows:

● 173;● 52, 51 TMT,

84 and 26.

Species are shown with different symbols: ●B. laxiflora, ♦B. excelsa, ■B. stenophylla,

▲B. bipinnata, ►B. grandifloia, ▼B. penicillata.



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HPLC (High Performance Liquid Chromatography)Analysis

Sophisticated analysis

Technique for the separation of complex mixtures

PumpDetectort

Gradient Controller

Injector

♦

Column

Solvents



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AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

B. bipinnata B. stenophylla

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

2.20

2.40

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

AU

0.00

0.10

0.20

0.30

0.40

0.50

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

AU

0.00

0.50

1.00

1.50

2.00

2.50

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

B. excelsa B. laxiflora

HPLC Analysis

B. penicillata

B. simaruba

B6

1

7, 8

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

A1

A1

1

4

67, 84 6 7, 8

B5

6

A1

12

A1

17, 8

4

A1

1B2

B3

B4

1 2 3 4 5 6 7 8

ID 3-epi-lupeol

lupeol 3-epi-ß-amyrin

lupenone 3-epi-α-amyrin

ß-amyrin α-amyrin/ ß-amyrone

α-amyrone

Ret. time(min)

26.5 27.6 28.2 28.7 29.4 29.9 31.3 32.1



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AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

B. stenophylla

Coupling HPLC analysis with PCA & LDA

B6

1

7, 8

A1

4

6

1

ID of the peak A4 A3 A2 A1 B1 B2 B3 B4 B5 B6 B7 B8

Ret. time (min) 8.9 10 11.4 12 16 16.5 17 18 19.4 20 20.6 24

ID T33epilupeol lupeol

3epißamyrin lupenone

3epi αamyrin

ßamyrin

αamyrin/ ßamyrone

αamyrone T4

Ret. time (min)

25.5 26.5 27.6 28.2 28.7 29.4 29.9 31.3 32.1 33.5

AU

0.00

1.00

2.00

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00

Archaeological Sample

1

7, 8

46



AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

A1

1 6

7, 84

B. bipinnata

43210-1-2-3-4

4

3

2

1

0

-1

-2

First Component

Seco

nd C

om

ponent

2 First PCA for HPLC data for 5 species

Distribution in the hyperspace of the first two components of PCA for 6 species according to their botanical origin.

Species are shown with different symbols:●B. laxiflora, ♦B. excelsa, ■B. stenophylla,▲B. bipinnata, ▼B. penicillata.



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Put into group B. bipinnata B. excelsa B. laxiflora B. penicillata B. stenophylla

B. bipinnata 10 0 0 0 0

B. excelsa 0 4 0 0 0

B. laxiflora 0 0 12 0 0

B. penicillata 0 0 0 6 0

B. stenophylla 0 0 0 0 8

Total N 10 4 12 6 8N correct 10 4 12 6 8Proportion 1 1 1 1 1

N = 40 N Correct = 40 Proportion Correct = 1. 000

Fitting matrix of the LDA Rows represent the true class, and the columns the assigned class.



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Put into group B. bipinnata B. excelsa B. laxiflora B. penicillata B. stenophylla

B. bipinnata 10 0 0 0 1

B. excelsa 0 3 0 0 0

B. laxiflora 0 1 12 0 0

B. penicillata 0 0 0 6 0

B. stenophylla 0 0 0 0 7

Total N 10 4 12 6 8

N correct 10 3 12 6 7

Proportion 1 0.75 1 1 0.875

N = 40 N Correct = 38 Correct Proportion = 0.950

Confusion matrix of LDA. Rows represent the true class, and the columns the assigned class. Wrong assignments are shown in red.



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Aplication to Archeological Samples

Species are shown with different symbols●B. laxiflora, ♦B. excelsa, ■B. stenophylla,▲B. bipinnata, ▼B. penicillata.

Archaelogical Samples

43210-1-2-3-4

5

4

3

2

1

0

-1

-2

-3

First Component

Second C

om

ponent

HPLC- PCA data with archeological samples



26-28 Juin 2012

Both chromatograms, and spectra differe according to species of the sampleChromatograms exhibit the different molecular composition of the resin according to its

origin Richer species in triterpens are B. bipinnata & B. stenophylla

Molecular composition of B. bipinnata & B. stenophylla is closer to molecular

composition of archeological samplesGlobal performance of PCA and LDA coupled to FTIR and HPLC in validation matrix was

close (95.2% and 95% respectively). HPLC-PCA exhibit an excellent discrimination power in the fitting matrix of 100% while

FTIR-PCA only reached 95.2%. FTIR-PCA & LDA exhibit 100% of discrimination capability for B.stenophylla samples,

while only 75% of B. bipinnata in both fitting and validation matrix. HPLC-PCA & LDA exhibit 87.5% of discrimination capability for B.stenophylla samples,

100% for B.bipinnata and 75% for B. excelsa in validation matrix.

Conclusions



26-28 Juin 2012

Coupling PCA and LDA to analytical technique allow us to establish a mathematical model to differentiate these resins

FTIR is the fastest tool and needs smaller amounts of material (g) which allow to analyze resins with lower triterpen content

HPLC exhibits a higher discrimination power of close species like B. bipinnata and B. stenophylla owing to its separation capacity

These results were completed by a GC-MS study

Molecular knowledge of these materials : - Establishing appropriate conservation/restoration plans, - Enlarge historical knowledge

Perspectives



26-28 Juin 2012

Thank You!

Paola [email protected]

Coupling multivariate statistical tools with analytical techniques for the study of copals in cultural heritage

Intensive European Spring School in Conservation Science

University of Avignon & Vaucluse

26-29 Juin 2012

PhD. Irma Belio : expert in Biomaterials, University of Sinaloa PhD. Lauro Bucio : expert in Biomaterials, National Autonomous University of MexicoPhD. Rito Vega : expert in Botanic University of Sinaloa PhD. Leonardo Lopez : director of Archaeological Project of Templo Mayor

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