Techniques of isolation and analysis in the natural product research

Sample preparation, isolation techniques Identification methods, structure elucidation Determination of absolute configuration

(stereochemistry) of natural products in microscale

Biological methods of testing activity of isolated natural products

Good knowledge of the life cycle of the selected organism

does it produce chemical signals? when does the production reach its maximum? which organ/tissue/gland produces the signal?

female moth calling

Sample preparation hydrodistillation (essential oils) solvent extraction (universal) „head-space“ techniques (volatile compounds) solid-phase microextraction, SPME (volatile

compounds) solid sample injection (insect glands)

Hydrodistillation, steam distillation

Clevenger apparatus plant material cut in pieces,

boils in water essential oil distil off together

with steam, forming the upper layer

Hydrodistillation, steam distillation

_____________________________________________________ Advantage Disadvantage _____________________________________________________ large scale possible danger of artefacts (oxidation)

suitable for plants, not insects

_____________________________________________________

Solvent extraction_____________________________________________________ Advantage Disadvantage _____________________________________________________ simple presence of balast compounds

possible to repeat pure solvents needed

analysis sometimes a low concentration

(amounts produced in the moment

of extraction)

_____________________________________________________

Supercritical fluid extraction fluid, which has the ability

of dissolution at the supercritical pressure and the supercritical temperature

most often used CO2

higher dissolving capability for various substances

extraction is fast

Supercritical fluid extraction_____________________________________________________ Advantage Disadvantage _____________________________________________________ good extraction potential expensive apparatus

room temperature CO2 is a greenhouse gas

reuse of solvent

CO2 - green chemistry

safe in food processing

cheap and easy to handle

large scale possible

_____________________________________________________

„Head-space“ techniques

static dynamic

Trapping volatiles:

sorbents: charcoal Porapak Q Tenax

elution with solvent freezing out

„Head-space“ techniques

„Head-space“ techniques___________________________________________________________________________

Advantage Disadvantage ___________________________________________________________________________

accurate composition apparatus needed

higher concentration pure solvents needed

compared to the extraction danger of „break-through“

possible to repeat danger of contamination

analysis of the loop

closed loop possible _____________________________________________________________________________

Codling moth (Cydia pomonella) gland 10:OH 12:OH E9-12:OH E8E10-12:Ald E8E10-12:Ac E8E10-12:OH Z8E10-12:OH E8Z10-12:OH 14:OH 16:OH 18:OH 18:Ac 20:Ac

head-space 10:OH 12:OH E9-12:OH - - E8E10-12:OH Z8E10-12:OH E8Z10-12:OH 14:OH 16:OH 18:OH - -

Solid phase microextraction SPME developed originally for trace analysis

of organic compounds in water adsorption on a thin film of polysiloxane thermal desorption in GC injector

SPME

Gas chromatography sensitive analytical technique potent in separation complex mixtures evaporation of the sample in a heated

injector capillary silica chromatographic

column (standard 30 m x 0.25 mm) siloxane bound to the column walls carrier gas – He, H2, N2 flame ionisation

detector or selective detectors

SPME_____________________________________________________________________

Advantage Disadvantage _____________________________________________________________________

without solvent only one analysis

high sensitivity equipment needed

simple

use in the field possible _____________________________________________________________________

Chromatograms - DHS and SPME

head-space

SPME

Small („light“) molecules discriminated (preference of „havier“molecules)

head-space

SPME

Solid sample injection

biological material (gland) sealed in a capillary

injection port adapted

vaporisation of volatiles in the injector directly

Solid sample injection__________________________________________________________________________________

Advantage Disadvantage __________________________________________________________________________________

without solvent only one analysis

no loss of compounds injector port specially adapted

cleaning of injector needed ___________________________________________________________________________________

Structure elucidation classical spectral methods used in organic

chemistry (IR, NMR, MS, UV, CD) – larger amount of sample (mg)

derivatisation, degradation, X-ray „hyphenated techniques“ (GC-MS, LC-MS,

GC-IR) – small amount of sample (µg) GCxGC-MS (2D-GC-MS, latest technique) 2D-GC determination of absolute

configuration (standards)

Stereochemistry double bond configuration (geometry)

absolute configuration (chirality, enantiomer, antipode)

(Z)-But-2-ene (cis)     

(E)-But-2-ene (trans)

                   

     

                   

(S)-Alanine (R)-alanine

Spectroscopy and wavelength

wavelength range from 2,500 to 16,000 nm vibrational excitation of covalently bonded atoms and

groups each functional group has a characteristic frequency

Infrared (IR) spectroscopy

IR spectrum of formaldehyde

Nuclear magnetic resonance spectroscopy (NMR)

excitation in magnetic field elements with odd number of protons + neutrons

(1H, 13C, 31P, 19F) each type has a characteristic frequency (chemical shift)

1H-NMR 2D-NMR

Ultraviolet (UV) spectroscopy range 200 to 800 nm unsaturated compounds absorb UV light typical absorption of funcional groups

Optical rotation, circular dichroism chiral compounds, polarised light CD = absorption in UV + optical rotation

Mass spectrometry Molecules in high vacuum Ion source, Electron ionisation Typical fragmentation of a molecule

mass spectrum of n-decane

Information from mass spectrum Molecular weight – from molecular ion M+.

Structure – from fragments (hard ionisation, MS/MS) Elemental composition – from exact mass

Gas chromatography - mass spectrometry (GC-MS)

benchtop instrument, usually without direct inlet

silica capillary column, inner diameter 0,25 mm, carrier gas helium

end of the column introduced into the ion source

2 classical types - quadrupole and ion trap new type –Time Of Flight (TOF)

Quadrupole GC-MS

obrázek

Quadrupole GC-MS

ions arise in ion source electron ionisation (EI) positive and negative ions possible to record chemical ionisation (CI) – determination

of molecular weight (reaction gas methane, ammonia, isobutane)

change from EI to CI is time-demanding in some instruments

10.000 20.000 30.000 40.000 50.000 60.000 70.000 80.000rt0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

20 40 60 80 100 120 140 160 180 200 220 240 260m/z0

100

%

88

554341

39 457370

60 8374

101

89 157115102 143129 183171

185 228199

88

43412927 39

5545

7361 7083

101

89183157143125102111 171

228185 199

Hit 3R:931 NIST 29645: DODECANOIC ACID, ETHYL ESTER

88

43

41

55

45

57

6960

7083

74

101

89

95 157115111 143129 171

Hit 4R:911 NIST 46382: NONADECANOIC ACID, ETHYL ESTER

88

4341

29

27 39

55

45

54

7357 70

67 8374

101

89 157143115102 129

197171158 185

199242213

Hit 5R:867 NIST 32384: ETHYL TRIDECANOATE

88

4341

27 3955 57 69

59 71 83

101

97 157143102 129111 228171 197185 199

Hit 6R:849 NIST 29640: UNDECANOIC ACID, 2,8-DIMETHYL-, METHYL ESTER

30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250m/z0

100

%

88

55

4341

3945

51

737057

60

67

83

81

101

89

9790

157

115102 143129139 144

183

171158

185 228199186

200 213 229

Quadrupole GC-MS

spectra comparable with big sector spectrometers

comparison with databases National Institute of Standards and Technology (over 60 000 spectra) and Wiley Library (230 000 spectra)

sensitivity can be increased using Selective Ion Monitoring (registeration of one fragment only)

Ion trap

Ion trap

classical - internal ionisation ions arise in the ion trap where they are

stored and analysed higher sensitivity than quadrupole GC-MS recorded spectra sometimes different from

sector spectrometers EI and CI possible in one injection tandem technique MS/MS and MS(n)

Comparison quadrupole – ion trap (selectivity of detection)

20.000 22 .000 24 .000 26.000 28 .000 30 .000 32 .000 34 .000rt0

100

%

1 7 1 8 1 9 2 0 2 1 m i n u t e s

quadrupole GC-MS

ion trap

Spectrum of hexadec-9-en-1-ol

50 100 150 200 250m/z

0%

25%

50%

75%

100%

3955

67

81

95

109

124138

152 166180 194

207

222

241

40 60 80 100 120 140 160 180 200 220 240m/z0

100

%

55

41

31

32

54

53

828167

56

66

69

70

9695

83

84

10997

98123

138 222152 166 194180 207 240224

ion trap

quadrupole GC-MS

Spectrum of hexadecan-1-ol

50 100 150 200 250m/z

0%

25%

50%

75%

100%

39

55

6983

97

111

125

138152 166 180 196 225

40 60 80 100 120 140 160 180 200 220 240m/z0

100

%

55

43

41

31 54

836957

68

67

58

7082

71

81

97

84

85

95

111

98

110125112

139 196154 168 181 224222

ion trap

quadrupole GC-MS

GC-TOF

Advantage of GC-TOF

mass determined at high resolution (elemental composition of ions, slow data collection)

two-dimensional chromatography possible, GCxGCxTOF (quick data collection, but nominal mass only)

OR

Mass spectrum does not differ at different parts of chromatograhic peak

Difference between scanning techniques and TOF

60 80 100 120 1400

250

500

750

1000

Rel

. Abu

nd.

m/z

60 80 100 120 1400

250

500

750

1000

Rel

. Abu

nd.

m/z

60 80 100 120 1400

250

500

750

1000

Rel

. Abu

nd.

m/z

42.5 43.0 43.50

2000

4000

6000

8000

Inte

nsity

Time (sec)

42.5 43.0 43.50

2000

4000

6000

8000In

tens

ity

Time (sec)

42.5 43.0 43.50

2000

4000

6000

8000

Inte

nsity

Time (sec)

60 80 100 120 1400

250

500

750

1000

Rel

. Abu

nd.

m/z

60 80 100 120 1400

250

500

750

1000

Rel

. Abu

nd.

m/z

60 80 100 120 1400

250

500

750

1000

Rel

. Abu

nd.

m/z

GC PeakSimultaneous Sampling Scanning

Chromatographic system

first dimension – classical column, non-polar, 30 m x 0.25 mm

secon dimension – short and narrow column (1-2 m x 0,1 mm), polar phase

separation on the second column is very fast, therefore data collection must be fast, too

Fast GC: short time of analysis Conventional GC Fast GC

naphtalene derivatives

187 compounds in 75 min 187 compounds in 5 min

TOF - fast data collection, very narrow peaks can be recorded

10 spectra/s

250 spectra/s

dodecane

150 ms peak width

2D-Technique removes the chemical noise and increases the sensitivity

9-d29-C16:COOEt

Coelution of analytes of very different concentration

propylene glycol

furfural

peak area of furfural is 0.001 % of the glycol peak area

35 Seconds

At Peak 45 Seconds

50 Seconds

Spectra recorded at different parts of the peak of propylene glycol

without deconvolution it is impossible to determine furfural

deconvolution

library spectrum

manual subtraction of spectra

Comparison of spectra

Liquid chromatography – mass spectrometry (LC-MS)

large amounts of mobile phase has to be removed

particle beam interface thermospray (TSP) electrospray (ESI) chemical ionisation in atmospheric

pressure (APCI)

LC-MS

only the technique of „particle beam interface“ gives spectra comparable with sector spectrometers

other techniques give quasimolecular ions (addition or elimination of particle from molecular ion)

GC-FTIR (Fourier transform infrared spectroscopy)

flow detection cell covered with a layer of gold („light pipe“)

less sensitive compared to GC-MS (~ 100x) GC columns of a larger inner diameter

(0,32-0,5 mm) carrier gas helium spectra in gas phase

(no intermolecular interactions)

Double bond configuration (E,Z)

3012 cm-1

OO

H H

Derivatisation in microscale characterisation of functional groups

in the molecule spectra easier to interpret better separation increased volatility or thermal stability

of compounds for GC analysis enantiomeric composition improvement of detection properties

Derivatisation reactions

methylation of acids with diazomethane CH2N2 (for GC)

acetylation of alcohols and amines (for GC)

silylation of alcohols (for higher volatility)

R

O

OHR

O

OCH3

ROH

RO

O

CH3

RNH2

R

HN

O

CH3

ROH

RO

Si

H3C

CH3

CH3

Derivatisation reactions dimethylhydrazones NH2N(CH3)2 (CO, CHO)

transesterification of triacylglycerols (for GC)

catalytic hydrogenation (carbon skeleton chromatography)

R O R NN

CH3

H H CH3

OCO

O

OCO

COmixture of FAME

Double bond position ozonolysis - pure compound only, formation of

aldehydes other oxidative cleavage of C=C bond (RuO4) –

formation of acids oxidation with OsO4 – formation of a diol methylthiolation of double bond (reaction with

dimethyl disulfide, DMDS, (CH3)2S2) – possible in mixtures

epoxidation with m-chlorperoxybenzoic acid (MCPBA) and following reactions

Double bond position

R2 CH

CH

R1CH3S SCH3

OHC R1

R2 CH

CH

R1

R2HC

HC R1

HO OH

R2HC

HC R1

O

R2HC

HC R1

Me3SiO OSiMe3

ClC6H4COOOH

[SiMe3]2NH

OsO4

O3

R2 CHO

Me2S2/I2

Fragmentation of DMDS adducts

m/z0

100

%

43

41

17361

55

8169

79

87171

12395122

231

174 232 404

m/z 173 m/z 231

(CH2)3

SS M+ 404

OAc(CH2)5

octadec-9-en-1-yl acetate

50 100 150 200 250 300 350 400m/z0

100

%

55

41

43

6781

79

11795

87 97 215123

171291269

338 386

(CH2)8

M+. 386

m/z 215

m/z 269

CHO

S S

SS

m/z 171

m/z 117

spectrum DMDS adducts of icosa-11,15-dienal

Chemical ionisation - methylvinylether

R1

HC

HC R2

H2C C OCH3

H

R1

HC

HC R2

H2C C OCH3H

R1

HC

HC R2

C CH2

R2

HC

HC OCH3 R1

HC

HC OCH3

H3COH

Chemical ionisation with acetonitrile in ion trap MS

100 150 200 250 300 m/z0%

25%

50%

75%

100%

97 111 123 137 151 165180

194 204 222 236250

265 279302

320

CI spectrum octadec-11-enal

active particle [C3H4N]+

m/z 54

CH3(CH2)4 (CH2)8CHO

m/z 250

m/z 180 M+320

[C3H4N]+

Absolute configuration of natural products

enantiomers may have different ecological functions

enantiomers may have different physiological effects

determination of enantiomeric purity of natural products is important

(S)-Alanine (R)-alanine

Different species of one genus use opposite enantiomers

HO HO

(+) (–)

ipsdienolIps paraconfusus Ips calligraphus

One enantiomer attracts males, the other one females

O

O

O

O

(R)-(–)-oleanemales

(S)-(+)-oleanefemales

fruit fly Dacus oleae, pest on olives

Biotransformation of resin components to the aggregation pheromone of bark beetles

OH

(–)--pinene (–)-cis-verbenol

(+)--pinene

OH

(+)-trans-verbenol

spruce bark beetle(Ips typographus)

Determination of the absolute configuration

separation and measurement of optical rotation

chiroptical methods NMR with shift reagents preparation of diastereoisomers enantioselective chromatographic

separation

Classical methods

large amount of pure natural product needed

separation of the enantiomeric pair from other sample components is difficult

measurement of optical rotation – inaccurate

Enantioselective chromatographic separations

columns based on cyklodextrin, hydroxy groups substituted with different groups

O

HO

OH

HO

O

O

HO OH

OH

O

O

HO

OH

HO

OO

OH

HO

OH

O

O

OHHO

HO

OO

O

OH

HO

-Cyclodextrin

OH

-cyclodextrin, 6 units-cyclodextrin, 7 units-cyclodextrin, 8 units

Two-dimensional GC

2D-GC, separation examples

Advantages of 2D-GC possible in minute quantities presaparation of components no needed high accuracy provided good (base-line)

separation of enantiomeric pairs high sensitivity, detection of minor

impurities of the opposite enantiomer information on enantiomeric purity of

several components in one analysis

standards needed!