techniques of isolation and analysis in the natural product research u sample preparation, isolation...
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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!
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