The effect of the solvent on the The effect of the solvent on the The effect of the solvent on the

The effect of the solvent on the

plasma degradation of phorbol plasma degradation of phorbol plasma degradation of phorbol

plasma degradation of phorbol

esters extracted from Jatropha oilesters extracted from Jatropha oilesters extracted from Jatropha oil

esters extracted from Jatropha oil

IC-PLANTS 2013(2013/2/2、Gero)

Osaka Prefecture UniversityH.Matsuura, S.Kongmany

M.Furuta, K.Imamura,Y.Maeda and S.Okuda

Background

• What is biodiesel?– It is an alternative for light oil,

which is extracted from seed of some kinds of plants.

• What is most useful to produce it?– Jatropha curcas (南洋脂桐) attracts

attention all over the world.• Why is detoxification needed?

– Jatropha oil contain phorbol esters (PE) that act a strong cancer promoter.

Why Why Why

Why

is degradation of J. curcas PEs needed?is degradation of J. curcas PEs needed?is degradation of J. curcas PEs needed?

is degradation of J. curcas PEs needed?

Present in Jatropha, Phorbol ester (PEs) is like double-edges sward. Present in Jatropha, Phorbol ester (PEs) is like double-edges sward. Present in Jatropha, Phorbol ester (PEs) is like double-edges sward.

Present in Jatropha, Phorbol ester (PEs) is like double-edges sward.

[Harinder P.S. Makkar et al. (2012)]

1 • Agricultural use: Natural molluscicide, fungicide, and bactericide against snails, fungal phytopahtogens and bacteria.

• Pharmacological tool: promoting cancer cells for cancer research.

2 • Toxic to human as tumor promoter and negative biological activities.Toxic to human as tumor promoter and negative biological activities.Toxic to human as tumor promoter and negative biological activities.

Toxic to human as tumor promoter and negative biological activities.

Biological Activity Biological Activity Biological Activity

Biological Activity

[Rakshit K. et al. (2007, 2011)]

• Action on Protein Kinase C, Tumor production, Platelet Aggregation, Cell Differentiation, and Metabolic Activities

ToxicityToxicityToxicity

Toxicity

[C-Y. Li et al. (2010)]

Pathological symptoms in lung and kidney, etc.Pathological symptoms in lung and kidney, etc.Pathological symptoms in lung and kidney, etc.

Pathological symptoms in lung and kidney, etc.

AnimalAnimalAnimal

Animal

100% Mortality (in days)100% Mortality (in days)100% Mortality (in days)

100% Mortality (in days)

mg PEs , consumed/kg body weightmg PEs , consumed/kg body weightmg PEs , consumed/kg body weight

mg PEs , consumed/kg body weight

Nubian goatNubian goatNubian goat

Nubian goat

7-97-97-9

7-9

10.5-13.510.5-13.510.5-13.5

10.5-13.5

Desert sheepDesert sheepDesert sheep

Desert sheep

3-53-53-5

3-5

4.5-7.54.5-7.54.5-7.5

4.5-7.5

Nubian goat kidNubian goat kidNubian goat kid

Nubian goat kid

7-117-117-11

7-11

10.5-16.510.5-16.510.5-16.5

10.5-16.5

LD50 = 27.34 mg/kg body weight in mice [Li. et. 2010]LD50 = 27.34 mg/kg body weight in mice [Li. et. 2010]LD50 = 27.34 mg/kg body weight in mice [Li. et. 2010]

LD50 = 27.34 mg/kg body weight in mice [Li. et. 2010]

What is superior of using plasma?

• Compact and low cost device to be used in developing countries

• no limitation such as radiation protection law

• many control knob to optimize process– working/environment gas, power, freq., so on

– dose/dose rate(radiation), freq./power(Sonic)

• easy suppression of individual promoter's effect(ion, UV, radical, heat)

Schematic drawing of plasma source

Heat influx from plasma is smaller than gas cooling.

Plasma Degradation of PEPlasma Degradation of PEPlasma Degradation of PE

Plasma Degradation of PE

sss

s

amplesamplesamples

amples

Plasma has a direct interaction with the solvent(water), which seems to become another electrode. Its temperature increase is, however, kept to be quite small.

Distance between sample and plasma source

Narrow gap(rich radical?) wide gap(no interaction)

Analytic Analytic Analytic

Analytic

InstrumentationInstrumentationInstrumentation

Instrumentation

ColumnColumnColumn

Column

Shim-pack XR-ODSII (2.0 mm i.d x 75 mm) Temperature : 35 oC.

Mobile PhaseMobile PhaseMobile Phase

Mobile Phase

Binary Gradient Mode: Pump A: water (H2O) Pump B: acetonitrile( ACN)Total flow: 0.400 mL

PDAPDAPDA

PDA

Scan mode: = 200 – 400 nm. Slit width : 1.2 nmLams : D2/WCell temperature: 40oC

Injector and analysis timeInjector and analysis timeInjector and analysis time

Injector and analysis time

Auto-sampler with 2 L injection volume.The phorbol esters detection was at =280nmLC stop time : 25 min.

Radiation Research CenterRadiation Research CenterRadiation Research Center

Radiation Research Center

Fig. 2 Fig. 2 Fig. 2

Fig. 2

LCMS 3080 equipped with both photodiode array detector (PDA) and Mass spectrometry detector (Shimadzu, Japan).

Calibration curveCalibration curveCalibration curve

Calibration curve

Fig. <Fig. <Fig. <

Fig. <

………

…

> > >

>

Calibration curve of standard phorbol ester using TPA (Phorbol-12-Myristate-13-Acetate)

and linear regression equation with relative coefficient of 0.9995

Fig. 3 Fig. 3 Fig. 3

Fig. 3

Preparation of TPA standard solution with concentration of 10, 30, 50, 70 and 100 ppm (mg/L).

Radiation Research CenterRadiation Research CenterRadiation Research Center

Radiation Research Center

Treatment of aqueous solution

HPLC/UV chromatograms of aqueous solution of: (a) HPLC/UV chromatograms of aqueous solution of: (a) HPLC/UV chromatograms of aqueous solution of: (a)

HPLC/UV chromatograms of aqueous solution of: (a)

TPA and (b) mixed jatropha phorbol esters (4 peaks each TPA and (b) mixed jatropha phorbol esters (4 peaks each TPA and (b) mixed jatropha phorbol esters (4 peaks each

TPA and (b) mixed jatropha phorbol esters (4 peaks each

peak corresponding to each type of jatropha factor) peak corresponding to each type of jatropha factor) peak corresponding to each type of jatropha factor)

peak corresponding to each type of jatropha factor)

before and after plasma irradiation for 15 min.before and after plasma irradiation for 15 min.before and after plasma irradiation for 15 min.

before and after plasma irradiation for 15 min.

Molecular structure of PE

Molecular structures of (a) 4ß-12-O-tetradecanoyl phorbol-13-acetate (TPA) and (b) jatropha phorbol esters (jatropha factor C1 to C6) found in jatropha curcas L.

Treatment of methanolic solution

HPLC/UV chromatograms of methanolic solution of: (a) TPA and (b) mixed jatropha phorbol esters (4 peaks each peak corresponding to each type of jatropha factor) before and after plasma irradiation for 15 min.

How How How

How

is the flow of J. curcas PEs during biodiesel production is the flow of J. curcas PEs during biodiesel production is the flow of J. curcas PEs during biodiesel production

is the flow of J. curcas PEs during biodiesel production

processes?processes?processes?

processes?

Sources: Sources: Sources:

Sources:

H. Makkar et al., Removal and Degradation of Phorbol Esters during Pre-treatment and Transesterification of H. Makkar et al., Removal and Degradation of Phorbol Esters during Pre-treatment and Transesterification of H. Makkar et al., Removal and Degradation of Phorbol Esters during Pre-treatment and Transesterification of

H. Makkar et al., Removal and Degradation of Phorbol Esters during Pre-treatment and Transesterification of

Jatropha curcas Oil, J Am Oil Chem Soc (2009) 86:173Jatropha curcas Oil, J Am Oil Chem Soc (2009) 86:173Jatropha curcas Oil, J Am Oil Chem Soc (2009) 86:173

Jatropha curcas Oil, J Am Oil Chem Soc (2009) 86:173

–––

–

181181181

181

J. J. J.

J.

Curcas Curcas Curcas

Curcas

seedseedseed

seed

Oil Oil Oil

Oil

extractioextractioextractio

extractio

nnn

n

Crude Crude Crude

Crude

OilOilOil

Oil

Oil Oil Oil

Oil

RefiningRefiningRefining

Refining

FertilizersFertilizersFertilizers

Fertilizers

Live-stock Live-stock Live-stock

Live-stock

feedingfeedingfeeding

feeding

DegummingDegummingDegumming

Degumming

Degummed oilDegummed oilDegummed oil

Degummed oil

BleachingBleachingBleaching

Bleaching

DeodorizationDeodorizationDeodorization

Deodorization

Refined OilRefined OilRefined Oil

Refined Oil

TransesterificationTransesterificationTransesterification

Transesterification

Crude Crude Crude

Crude

BiodieselBiodieselBiodiesel

Biodiesel

Glycerol & ImpuritiesGlycerol & ImpuritiesGlycerol & Impurities

Glycerol & Impurities

Washing Washing Washing

Washing

with waterwith waterwith water

with water

Waste Waste Waste

Waste

waterwaterwater

water

Clean Clean Clean

Clean

BiodieselBiodieselBiodiesel

Biodiesel

DeacidificationDeacidificationDeacidification

Deacidification

Acid gums and wastewaterAcid gums and wastewaterAcid gums and wastewater

Acid gums and wastewater

Seed Seed Seed

Seed

cakecakecake

cake

• 70-75% PEs 70-75% PEs 70-75% PEs

70-75% PEs

move to the oil.move to the oil.move to the oil.

move to the oil.

• 25-30% PEs 25-30% PEs 25-30% PEs

25-30% PEs

retain in the seed retain in the seed retain in the seed

retain in the seed

cake.cake.cake.

cake.

• About 22.58% and 3.98% of PEs were reduced after degumming step from crude oil obtained by solvent extraction and pressing, respectively.

PEs are completely degraded at deodorization at temperature 260oC under reduced pressure.

Direct transesterification of oil, PEs would be converted into original phorbol contaminating into the glycerol phase.

Phorbol ester content (expressed in mg/g Phorbol ester content (expressed in mg/g Phorbol ester content (expressed in mg/g

Phorbol ester content (expressed in mg/g

±±±

±

SD, n = 3) of the different fractions obtained SD, n = 3) of the different fractions obtained SD, n = 3) of the different fractions obtained

SD, n = 3) of the different fractions obtained

during pre-treatment and transesterification of three different Jatropha oil samples.during pre-treatment and transesterification of three different Jatropha oil samples.during pre-treatment and transesterification of three different Jatropha oil samples.

during pre-treatment and transesterification of three different Jatropha oil samples.

ParametersParametersParameters

Parameters

Solvent extractedSolvent extractedSolvent extracted

Solvent extracted

PressedPressedPressed

Pressed

Non-toxicNon-toxicNon-toxic

Non-toxic

Crude oil 3.10 ± 0.25 3.77 ± 0.03 ND

Degummed oil 2.48 ± 0.24 3.62 ± 0.19 ND

Acid gums 2.02 ± 0.07 3.35 ± 0.00 ND

Wash water 2.72 ± 0.01 2.08 ± 0.48 ND

Silica-treated oil 2.51 ± 0.33 3.76 ± 0.50 ND

Spent silica NA NA NA

Stripped oil ND ND ND

Fatty acid distillate ND ND ND

Biodiesel ND ND ND

Crude glycerine ND ND ND

Biodiesel wash water ND ND ND

ND: Not detectable; NA: Not analyzed

Sources: Sources: Sources:

Sources:

H. Makkar et al., Removal and Degradation of Phorbol Esters during Pre-treatment and Transesterification of H. Makkar et al., Removal and Degradation of Phorbol Esters during Pre-treatment and Transesterification of H. Makkar et al., Removal and Degradation of Phorbol Esters during Pre-treatment and Transesterification of

H. Makkar et al., Removal and Degradation of Phorbol Esters during Pre-treatment and Transesterification of

Jatropha curcas Oil, J Am Oil Chem Soc (2009) 86:173Jatropha curcas Oil, J Am Oil Chem Soc (2009) 86:173Jatropha curcas Oil, J Am Oil Chem Soc (2009) 86:173

Jatropha curcas Oil, J Am Oil Chem Soc (2009) 86:173

–––

–

181181181

181

HowHowHow

How

is the flow of J. curcas PEs during biodiesel production processes?is the flow of J. curcas PEs during biodiesel production processes?is the flow of J. curcas PEs during biodiesel production processes?

is the flow of J. curcas PEs during biodiesel production processes?

Conclusion

• UV Chromatogram is successfully applied to quantitative analysis of Phorbol Esters in Jatropha oils, and degradation of Phorbol ester with atmospheric plasma and is confirmed.

• Effect of plasma irradiation depends not only on the molecular structure of PE, but also on the kind of solvent.

• This may be due to production process of active radical such as OH.

Future works• Test using PE with much simpler structure to

study degradation mechanism.

• Plasma diagnostic and radical measurement, aiming intelligent plasma processing • Solvent temperature( -->SPP30 ) • OH concentration( -->ISPlasma2013 )• Input energy (discharge current)• plasma density and temperature

• Direct confirm of detoxification, not merely disappear of Phorbol Ester

Appendix

BackgroundAtmospheric pressure plasma is now attractive for decontamination of environmental waste and medical technology, because of

low cast without vacuum pumping systemapplicability for biomaterial that does not less tolerate vacuumlarge process speed

Recently application of plasma to solution samples has gathered much attention in chemical of food industry field. (ex. degradation of Phorbol esters)

Temperature monitor of treated samples is importantto keep treatment process.to estimate heat influx and deduce physical processes with it.

Preliminary heat flux estimation on solid target has already reported.But measurement on liquid target is found to have a problem.

Degradation of Phorbol Ester

OC

CH2OH

H

OH

H3C

O

H

OH

H3C

CH3CH3

H

CCH3

H3C(H2C)11H2CC

O

O

O

US / US / US /

US /

γ-ray-ray-ray

-ray

2 (•OH) 2 (•H)

TetraDecanoyl Phorbol-13-Acetae (TPA)TetraDecanoyl Phorbol-13-Acetae (TPA)TetraDecanoyl Phorbol-13-Acetae (TPA)

TetraDecanoyl Phorbol-13-Acetae (TPA)

Phorbol 12-myristate 13-acetate (PMA)Phorbol 12-myristate 13-acetate (PMA)Phorbol 12-myristate 13-acetate (PMA)

Phorbol 12-myristate 13-acetate (PMA)

HOHO

CH2OH

H

OH

H3C

O

H

OH

H3C

CH3CH3

phorbol

H

PhorbolPhorbolPhorbol

Phorbol

S.Kongmany, 11th APCPST (Kyoto, 2012) 3-P92.

Estimate plasma heat flux

0

4

8

12

16

20

1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000

TC(center)

time[s]

TC[d

eg.]

datafitting

Estimation with exponential fitting Q（~dT/t) = 0.5 [W]

12

4

0

0.5 0.17

Target moved

Heat flux depends upon measurement position.

dTdecay time t is obtained by fitting.

Heat flux measurement with Cu target

Heat flux determination

Type-T TC Temperature gradient methodType-K TC Fitting/cut method

Electrically isolated

Easy to modify for Q-V characteristic

Material: Cu, Mo

provided by Osakabe(NIFS)

Yambe's work on calorimetry

He gas He plasma

Water temperature is monitored for He gas/plasma irradiation.(22P039-P, plasma conference 2011, Kanazawa)

The difference of two case corresponds to plasma heat flux.

They said 3.81E-5[J] energy was carried by 13[kHz] micro pulse, which consists plasma jet. Average heat flux is 0.5[W].

Is the thermal of water vessel isolation sufficient?

Is temperature in water homogeneous?

But thermocouple shows irregular behaviour, and can not be used.

Abnormal jump of TC signal Water(after 1000[s])

Reference(RT)

Bekar(bottom) recorded with mv100

When TC is in the water, TC signal jumps abnormally. Why?

Principle of thermocouple

TC wiring consists an large loop, which could act as an antenna, and the large induction current may flow.

Electrical cold junction

CT1200D (CUSTOM)

MV100 (YOKOGAWA)

ENET9211 (NI)

Tolerance of compensation circuit against large current noise is different depending on the data logger used.

Experiment setup

TC signals recorded with ENET9211

Room Temp.Baker wallWater by TC with sheathWater by bear TCWater by alcohol thermometer

Comparison of thermometer data

TC signals recorded with ENET9211

Room Temp.Baker wallWater by TC with sheathWater by bear TCWater by alcohol thermometer

Consideration on TC jump

• It occurs only when one TC is in water.• It has threshold discharge power.

– Even before breakdown, it may occur. – Even discharge continue, signal recovers from

it with reducing power.• It occurs for all TC simultaneously,

regardless measuring points.

This TC signal jump seems to the results of malfunction of cold-junction compensation circuit, not of interaction between TC material and radicals in water.

How to avoid

• Set TC outside baker (with isolation blanket)

• Use other kind of thermometer (such as Pt resistor thermometer)

• Use TC with noise shield or recorder with pulse isolation function

These methods would be tested in near future.

Conclusion

• Thermocouple temperature measurement often suffers signal jump and monitoring becomes impossible.

• This is not due to chemical process in water. Most possible cause is induction current in TC circuit.

• By reducing induction effect or by isolation the circuit, this problem might be overcome.

Atmospheric Pressure Plasma Jet

Power supplyLHV-13AC(Logy Electric Co.LTD.) Input AC100V/2A Output 10KV/120mA RMS 9～12KHz Size 140×92×175 Weight 1.4kg