Musetel Div

Copyright 2005 Comunicat CRC, LLC
Page 2
10.1 Introducere
testelor analitice a principiilor active de musetel are urmtoarele obiective
principale:
1. dovada calitativ relevante principii active farmacologic n Flo Matricariae s
2. Impactul i genetice factori ecologici pe compoziiei calitative i cantitative
a principiilor active
3. Elucidarea de biosintez a principiilor active individuale de musetel
Alegerea metodei analitice depinde de natura analizei. Sub toate circum-
circumstane, metoda aleas ar trebui s ofere acurateea necesar i
reproductibilitatea. Pentru
dezvoltarea unei analize de rutin, factori economici cum ar fi timpul i costurile
trebuie s fie luate n considerare.
10.1.1 T
EST
R
EGULATIONS N
P
HARMACOPOEIAS
Farmacopeile (Tabelul 10.1) furnizeaz, fizic, i cromatografice de testare
procedee chimice
pentru a detecta identitatea i puritatea de ulei esenial, precum i coninutul de
matricin.
Tabelul 10.1
Regulamentele de testare pentru flori de mueel i Preparate de musetel n
farmacopei
ncepnd cu 1882
An
Farmacopee
Regulamentele
1882
DAB (Deutsches Arzneibuch) (Farmacopeea german), dou
ediie
Nici un test de lege
1893
Pharmacopoea Helvetica, ediia a 3-
1894
DAB (Farmacopeea german), ediia a 3-
1897
Supliment la DAB 3, i anume, drogurile nu cele care sunt incluse n DAB
3, 2nd Edition
1900
DAB (Farmacopeea german), ediia a patra
1901
Svenska Farmakopen 8
1905
Farmacopeea Statelor Unite ale Americii, 1900 ediie
1905
Pharmacopoea Nederlandica 4
1905
Farmacopea Espaola 7
1906
Treilea Supliment la DAB 4
1906
Pharmacopoea Belgicae 3
Densitate, solubilitatea
1906
Pharmacopoea Austria 8
100 mercis ne minus Qam Ducate contradictorie 15
extracti spirituosi praebeant
1907
Farmacopeea din Japonia, ediia englez
1907
Pharmacopoea Helvetica 4
Densitate, coerena pe de rcire
1908
Pharmacope Franaise
1910
DAB (Farmacopeea german), ediia a 5-
1916
Supliment patrulea la DAB 5
1926
DAB (Farmacopeea german), editia a sasea
Determinarea coninutului de ulei esenial
1940
Pharmacopoea Nederlandica 5 (dou de imprimare)
1941
Supliment pentru DAB 6
Nu reglementri suplimentare pentru DAB 6
1941
Densitate, solubilitate, rotaia specific
1948
Pharmacopoea Danica
1958
Pharmacopoea Nederlandica 6
Determinarea coninutului de ulei esenial rezultate
n druppeltjes Blauwe
1960
sterreichisches Arzneibuch (Pharmacopoea Austria) 9
Determinarea coninutului de ulei esenial,
Identificarea prin reacia cu
dimetilaminobenzaldehida
TF4015_C010.fm Pagina 222 vineri 2005-04-opt 08:25

Pagina 3
Tabelul 10.1
farmacopei
ncepnd cu 1882 (continuare)
An
Farmacopee
Regulamentele
1971
Determinarea coninutului de ulei esenial,
factor de umflare, extract fluid, reacie de culoare,
valoarea pH-ului, coninutul de ulei esenial
1968
DAB (Farmacopeea al Republicii Federale Germania), 7
ediie
Determinarea coninutului de ulei esenial i
compararea culorii albastru
1964 /
1975
Farmacopeea din Republica Democrat German, 7-a ediie
Identificare prin reacie cu
dimetilaminobenzaldehida, n strat subire
cromatografie, absorbana de xilen
soluie de abur distilat la 600 nm
(Standard: guaiazulena soluie)
1975
Farmacopeea european, vol. III
Determinarea coninutului de ulei esenial, de test
reacie cu dimetilaminobenzaldehida,
cromatografie n strat subire
1976
Pharmacope Franaise 9
Determinarea coninutului de ulei esenial, care
trebuie s fie de o culoare albastru nchis
1980
British Farmacopeea
Determinarea coninutului de ulei esenial
1982
nregistrare Standard 36 AMG 76 (Germania)
Determinarea coninutului de ulei esenial, de test
reacie cu dimetilaminobenzaldehida,
cromatografie n strat subire
1987
DAB (Farmacopeea al Republicii Federale Germania), 9
ediie
o Determinarea) a coninutului de ulei esenial
b) Identificare prin reacie cu
c) de testare pe puritate de strat subire
cromatografie
1987
Pharmacopoea Helvetica VII, supliment 1993, Flos i Extr.
Lichid
c) Identificarea de strat subire
cromatografie, determinarea eseniale
coninut de ulei, gravimetrice
1990
Pharmacopoea Austria, Flos
cromatografie
1991
DAB (Farmacopeea german), ediia a 10-a, supliment 1993,
Flos
cromatografie
1997
DAB (Farmacopeea german) 1997, Extr. Fluid
a) de identificare prin strat subire
cromatografie
b) Determinarea coninutului de ulei esenial,
gravimetrice
1997
DAB 1997 (Farmacopeea german) 1997, Matricariae
aetheroleum
cromatografie
b) test puritate de profil cromatografic
TF4015_C010.fm Page 223 vineri 2005-04-opt 08:25

Pagina 4
Desigur, exist i alte numeroase publicaii pe analiza a principiilor active ale
musetel [1-10, 17-24, 27-31, 33, 36, 39-49, 52-66, 68-81, 83-89, 92-119, 123, 127,
130,
132-139, 141-143, 148-157, 159, 160, 162, 163]. Un rezumat i a metodelor de
evaluare a
fost publicate i actualizate continuu de Schilcher [116-121].
Cele mai recente descoperiri, precum i procedurile de testare stabilite n vrst
sunt obiectul prezentei
rezumat.
10.2 ANALIZA ale uleiului esenial
10.2.1 M
ETHODS DE
E
XTRACTION
Dou metode diferite - o distilare cu vapori aa cum este descris de Schilcher
( Tabelul 10.3 ) i un
clorura de metilen folosind extracie, n-hexan, i eter de petrol (la 40-60C) - au fost
comparate. Acesta din urm a dus la un randament mai mare de ulei esenial i
diferene semnificative n
coninut de spiroethers i de oxid de bisabolol A, precum i de oxid de bisabolol B,
bisabolol, bisabolone
oxid, i spathulenol.
Concomitent cu coninut ridicat de ulei esential, suma de compui hidrofobe
cum ar fi acizii grai i carotenoidelor a fost majorat. Cu toate acestea, acest lucru
nu va influena analiza de
cromatografie n faz gazoas (GC) sau (cromatografie n strat subire (TLC). loc
de cuantificare chamazu-
Lene - care lipsete n clorur de metilen extract - suma de matricin poate fi
determinat utiliznd TLC sau GC. La extragerea cu clorura de metilen, suma de
spiroethers
sa dovedit a fi majorat cu 68% n medie fa de distilare cu abur, oxid de bisabolol
A
cu 42%, oxid de bisabolol B cu 20%, bisabolol cu 12%, i spathulenol cu
12%. Rezultate similare
Tabelul 10.1
farmacopei
ncepnd cu 1882 (continuare)
An
, Farmacopeea
Regulamentele
1997 /
2002
Farmacopeea european, Flos
cromatografie
1997 /
2002
Farmacopeea European, Extr. Fluid
cromatografie
gravimetrice
2002
Farmacopeea european, Flos
b) Determinarea total apigenina-7-
glucoside prin cromatografie lichid,
cromatografie
2002
Farmacopeea European, Extr. Fluid
cromatografie
gravimetrice
2002
Proiect european monografia farmacopeei, Matricariae
aetheroleum
cromatografie
b) test puritate de gaz cromatografic profil

Page 5
au fost publicate [31, 42, 91]. n scopul de a stabili valoarea exact a anumitor
componente,
un timp de extracie de o or folosind clorura de metilen este recomandat.
Fost datele publicate rmn n continuare util pentru comparaii. Cele mai multe
dintre ele au fost obinute prin
distilare cu abur. Pentru investigaii calitative, metoda TAS, o metoda termica
dezvoltat de
E. Stahl [138], pot fi aplicate. Este foarte sensibil, astfel, un eantion mic (de
exemplu, trei capete de flori)
este suficient [119]. Un alt avantaj al metodei TAS este reducerea timpului necesar
pentru
analiz, care permite prelucrarea unei serii de probe n scurt timp. Etichetarea
experimente
sunt, de asemenea, posibil cu ajutorul metodei de TAS [119]. Cu toate acestea,
analiza se limiteaz la principalele
componente ale analizei.
Tabelul 10.2
Unele farmacopei conin Monografii pe flori de mueel Roman, Roman
Ulei de musetel sau Roman Preparate musetel (de exemplu, Extracte)
An
Farmacopee
Metode
1906
Pharmacope Belge 3
1908
Pharmacope Franaise
1934
HAB - Dr. Willmar Schwabe
1940
Nederlandsche Pharmacopee, ediia a cincea (olandez Ph)
1941
Farmacopeea Helvetica, ediia a 5-
1954
oficial Farmacopea Espaola IX (Hisp IX)
1980
British Farmacopeea
1953
Pharmacope Belge V
Flori, ulei volatil, apa musetel
1954
British farmaceutice Codex
ID: macroscop microscop.,.
1959
Farmacopeia dos Estados Unidos do Brasil
1960
Arzneibuch sterreichisches (OAB 9), Austria
Farmacopee
ID: macroscop.
: Ulei: Compoziie esenial distilare cu abur
1954
Farmakopea Polska III (polonez Ph)
1972
fusese declarat Farmacopea della Repubblica Italiana VIII
1976
British plante Farmacopeea
ID: a. macroscop., b. microscop., c. TLC
1976
Pharmacope Franaise IX
1979
Farmacopee Helvetica VI
1980
British Farmacopeea
ID: TLC
1981
sterreichisches Arzneibuch 1981 (Farmacopeea austriac
1981)
1983
British plante Farmacopeea
ID: macroscop,., TLC microscop.
1987 -
1993
Pharmacope Franaise X
1987
Pharmacopea Helvetica VII
1988
British Farmacopeea
ID: TLC
1992
DAB 10, german Farmacopeea ediia a zecea
1993
Farmacopee Helvetica VII
1993
British Farmacopeea
1997
Farmacopeea European, ediia a 3-
ID: TLC
1999
British Farmacopeea
2003
Farmacopeea european 4.3 (versiunea german)

Page 6
Tabelul 10.3
Determinarea cantitativ a de ulei esenial n flori de musetel, prin intermediul
Steam
Distilarea [112121]
Regulamentul
Test poriune
i gradul de
strivire
Distilarea /
menstruum
Solvent n
gradat
tub
Viteza de
distilare
Timp de
distilare
Timp de
lectur dup
distilare
Pharmacopoea
Austria, nou
ediie, 1960
20.0 g
nemcinat
400 ml de ap
Decalin
Se las s fiarb
3-4 h
5 min
Farmacopee de
german
Democratice
Republica, 7
ediie (DAB 7,
DDR), 1964 i
1975
10.0 g
nemcinat
135 ml de etilen
glicol, 15 ml de
ap, 0,2 g de
ulei de silicon
emulsie
-
Nu sunt specificate;
temperatura la
condensator nu
mai mare dect
25C
3 h
5 min
British Pharma-
copoeia, 1968
Nici o instructiune 300 ml de ap
Xilen
Se las s fiarb
astfel nct
partea de jos a
condensator
rmne rece
3-5 h
5 min
Farmacopee de
Federal
Republica
Germania, 7
ediie, 1968
DAB 7, BRD,
1968
25.0 g de
aspru
pulbere
300 ml de ap
Xilen
Se las s fiarb
2 h
15 min
Pharmacopoea
Helvetica, 6
ediie, 1971
5,0 g; gradul de
strivire nu
specificate
500 ml de ap, apoi extract cu pentan; determinare gravimetric similar cu DAB 6
European
Farmacopee -
proiectul din iunie
10, 1971
50.0 g
nemcinat
500 ml de 0,5 N
HCI
Xilen
3-4 ml / min.
4 h
10 min
Farmacopee
CISR, Ph.Bs. III,
vol. I
10.0 g, prin
sit 3
300 ml de ap
Decalin
Se las s fiarb
+ 4 h 30 min
5 min
Propunere H.
Schilcher [112]
10.0 g
nemcinat
resp. trecut
prin sit
Nr. 3
omogen
amestec
300 ml de ap
-
Coperta
"Pilz" "etapa
II, 220 volti,
300 watt =
aproximativ 4-5
ml / min =
aproximativ 40-45
picturi / min
Condensator s fie
oprit
dup 3 ore i
distilare care urmeaz s fie
a continuat pentru
aproximativ 5 min
pn cnd uleiul este
eliminate din
condensator de perete
15 min
European
Farmacopee,
vol. III, 1975
50.0 g
nemcinat
500 ml de 1%
Soluie de NaCl n
un balon cotat de 1000 ml
Xilen (1.0
ml)
3-4 ml / min
4 h
10 min

Page 7
10.2.2 V
OLUMETRIC I
G
RAVIMETRIC
D
ETERMINATION A
T
Otal
C
CONINUTUL
Determinarea gravimetric sa dovedit a da rezultate mai bune dect determinarea
volumetric
[120, 121].
Cuantificarea volumetrice a fost efectuat n conformitate cu liniile directoare ale
DAB (german
Farmacopeea) i a dat urmtoarele rezultate:
x = 0,57%
s = 7.572 x 10
-2
vc = 13.28% (% vc = deviaia standard relativ)
Tabelul 10.3
Determinarea cantitativ a de ulei esenial n flori de musetel, prin intermediul
Steam
Distilare (continuare)
Regulamentul
Test poriune
i gradul de
strivire
Distilarea /
menstruum
Solvent n
gradat
tub
Viteza de
distilare
Timp de
distilare
Timp de
lectur dup
distilare
Farmacopee de
Federal
Republica
Germania, 9
ediie, 1987
DAB 9, FRG,
1987
30.0 g
nemcinat
300 ml de ap n
un balon cotat de 1000 ml
Xilen
(0,5 ml)
3-4 ml / min
4 h
10 min
Pharmacopoea
Helvetica, 7
ediie, 1987
30.0 g
nemcinat
300 ml de ap n
un 1-L balon
Xilen
(0,5 ml)
3-4 ml / min
4 h
10 min
Pharmacopoea
Austria, 1990
30.0 g
nemcinat
300 ml de ap n
un 1-L balon
Xilen
(0,5 ml)
3-4 ml / min
4 h
10 min
German Pharmac.
DAB = 10
30.0 g
nemcinat
300 ml de ap n
un 1-L balon
Xilen
(0,5 ml)
3-4 ml / min
4 h
10 min
Eur Ph 1996.,, 3
ediie
30.0 g
nemcinat
300 ml de ap n
un 1-L balon
Xilen
(0,5 ml)
3-4 ml / min
4 h
10 min
Eur Ph.,
1997/2002
30.0 g
nemcinat
300 ml de ap n
un 1-L balon
Xilen
(0,5 ml)
3-4 ml / min
4 h
> 10 min
Eur Ph., 2002
Proiect de monografie
30.0 g
nemcinat
300 ml de ap n
un 1-L balon
Xilen
(0,5 ml)
3-4 ml / min
4 h
> 10 min;
rcire
ntrerupt
spre sfritul
de
distilare
Not: Din 1987, Parlamentul European, monografiile naional pe flori de musetel
au fost armonizate. n 1997, monografie
"Flos Matricariae" din Farmacopeea European ediia a treia a nlocuit monografii
naional i este acceptat oficial
n toate statele membre, din Farmacopeea Convenia European (31 de state n
2003), inclusiv cele 15 de state membre ale European
Uniunii Europene.
Copyright 2005 CRC Press, LLC

Page 8
Folosind acelai material planta, determinarea gravimetric a dat urmtoarele
rezultate:
x = 0.814%
s = 1.338 x 10
-2
vc = 1,64% (% vc = deviaia standard relativ)
Cu t-valoare (testul Student), calculat la 10.05, o comparaie a celor dou metode
nu este
posibil. Acest lucru explic gam larg de valori gsite n literatura de
specialitate. Volumetric Determinarea
este metoda oficial a Ph Eur., Dar metoda gravimetric se poate aplica la
eantioane mici.
10.2.3 T
HIN
-L
Ayer
C
HROMATOGRAPHY
ntr-o comparaie a cromatografie n strat subire-diferite condiii, utilizarea de
silicagel plci
(GF
254
) Ca faz staionar i o faz mobil de benzen / acetat de etil (95:5, V / V) a fost
gsit
da cele mai bune rezultate [109]. Benzenul poate fi nlocuit de ctre toxice toluenul
mai puin, fr pierderi n
separare de calitate. Solventul Acelai sistem este utilizat pentru analiza de ulei de
musetel si musetel
extract lichid n conformitate cu Farmacopeea german 1997. Alternativ, clorur de
metilen / etil
acetat (98:2, V / V) [101] i toluen / acetat de etil (93:7) [158] pot fi utilizate cu
rezultate bune. The
Farmacopeea European 1975, volumul III, recomandat cloroform / benzen
(75:25, V / V), care
nu respect standardele de concentrare maxima de lucru (MWC) i standard tehnic
Concentrare (TSC), dar alegerea a solvenilor o-la-la data metoda up ar trebui s
convin cu aceste
cerine (Tabelul 10.4). O faz foarte simplu mobil, cloroform pur, este specificat de
ctre European
Farmacopeea 1997, pentru identificarea de flori de musetel de TLC [161].
Sistemului de solvent ar trebui s fie alese n funcie de polaritatea
compuilor. Pentru substanele
de obicei, rmase aproape de linia de start (de exemplu, matricin), o faz mobil de
toluen / acetat de etil
(80:20, V / V) ofer Timpii reteniei utile (de exemplu, matricin: RF 0.13). Pentru
separarea bisabolol
oxizi, TLC plci silanizat poate fi folosit ca faza staionar n combinaie cu 0,1%
sau mai bine
0,2% acid acetic glacial 50% n toluen [36], faza mobil. Exist mai multe moduri
pentru a detecta
compui separate. Cea mai buna alegere este o combinaie ntre un SbCl
3
solutionand reactivul PE
[109]; alternativ, aldehid anason / acid sulfuric [149] sau vanilin / acid sulfuric
[158] ca spray
reactivi de munc bine.
Tabelul 10.4
x R
F
Valorile elementelor constitutive de musetel cu diverse faze Mobil
Constitutiv
Benzen
(Sau toluen) 95
Acetat de etil 5
(V / v)
Diclormetan 98
Etil acetat de 2
(V / v)
Cloroform 75
Benzen
sau toluen 25
(V / v)
x R
F
dup dezvoltare de peste 12 cm
tovo|ovcocvc
0.72
0.81
0.72
Chamazulene
0.68
0.78
0.69
cis-En-yne-dicycloether
0.46
0.71
0.46
trans-En-yne-dicycloether
0.42
0.68
0.42
Bisabolone oxid
0.38
0.64
0.39
Bisabolol
0.30
0.51
0.33
Spathulenol
0.21
0.42
0.24
Herniarin
0.19
0.32
0.21
Oxid de bisabolol A
0.18
0.30
0.19
Oxid de bisabolol B
0.13
0.27
0.16
Umbelliferon
0.02
0.04
0.02
Matricin
0.00
0.06
0.03

Page 9
10.2.4 D
ENSITOMETRY
Chamazulene i oxizi de bisabolol au fost cuantificate prin extragerea petelor lor de
pe placa pentru TLC
urmat de determinare fotometrice [109]. Msurarea direct a componentelor
principale pe TLC
a fost dezvoltat folosind o Shimadzu CS-900 sistem de scanare [120]. Spoturile au
fost scanate n
zig-zag modul la dou lungimi de und diferite. nlimea vrfului de semnal a fost
transformat ales
pentru cuantificarea (Figura 10.1).
Dup ce a recunoscut de acid carboxilic chamazulene (CCA), ca o profen natural,
grupul
of Imming studiat extensiv chimice i aspectele farmacologice ale acestui
compus. Goeters
[32] a constatat cromatografice urmtorii parametri s fie potrivite att pentru
preparare i
scopuri analitice: gel de siliciu, hexan / acetat de etil / metanol 65:30:5, RF (CCA)
0.5. Ea cuantificate
acid carboxilic chamazulene densitometric atunci cnd ea a privit pentru condiii
care cea mai mare a cedat
Coninutul CCA n perfuzii apos;-min extracie 8 la 100C a dat cele mai bune
rezultate (0.4 - 1.0 mg / ml
utiliznd 10 g de flori de cultivar "Mabamille"). Chamazulene nu a fost detectat n
perfuzie;
matricin a fost prezent, dar nu a fost cuantificat.
Coninutul en-yne-dicycloethers poate fi determinat cu precizie de nalt presiune
lichid
cromatografie (HPLC) sau prin densitometrie. Valorile de determinare
densitometric au fost
20-35 cromatografie% mai mare dect cele obinute de gaz de. Publicat folosind
metode de fotometrie
pentru a determina bisabolol i-en-yne dicycloethers [44] i densitometrie s
cuantifice bisabolol [155,
156] nu au fost reproductibile [44, 155, 156]. O metoda colorimetric pentru a
determina chamazulene
[152] au artat o mbuntire pic peste o metod publicat anterior [109].
Analiza GC a artat avantaje n ceea ce privete separarea de oxizi de bisabolol A,
B, C, Chama-
zulene, i farnesene i, prin urmare, este recomandat n loc de TLC.
Figura 10.1 TLC scanare de bisabolol, bisabolone, i oxizi de bisabolol. Scanner
Model: CS Shimadzu-900
Cromatografice-Scanner. (A) Se introduce placa pentru TLC n saturate SbCl
3
soluie pentru 5 sec; (b) se usuc la 110C timp de 5 min;
(C) se scufund n reactiv PE (acid acetic / acid fosforic) pentru 5 sec; (d) uscat n
curent de aer rece pn miros de
acid acetic nu mai este detectabil; (e) se nclzete pn la 110C timp de 10 min; (f)
de scanare imediat. Msurare lungime de und
pentru bisabolol i bisabolone (pete violet): 530 nm; lungime de und de msurare
de oxizi de bisabolol (pete roii):
520 nm, lungimea de und de referin pentru toate: 700 nm.
5
3.5
Bisabolon
Bisabolol

Pagina 10
10.2.5 R
EACTION
C
HROMATOGRAPHY
Degradarea de matricin a chamazulene pot fi detectate n timp ce ruleaz
TLC. Experimentele au
artat c matricin din extractele de tratate cu abur pentru 5-10 min este treptat
transformat n Chama-
i trei intermediari zulene care a dat trei pete albastre, dup separare TLC (a se
vedea figura 4.22 n
De referin 121).
10.2.6 G
AS
C
HROMATOGRAPHY
Cromatografice gaz prima separare a componentelor din uleiul esenial a fost
publicat n
1968 [49]. De atunci, GC mai multe metode au fost dezvoltate [16, 29, 31, 43, 44,
47, 49, 72,
74, 112, 113, 155, 156]. Att i nepolare coloane polare au fost folosite; de
exemplu, 5-10% Carbowax 20
M, 10% UCCW 982; 3% OV 17; 5%, SE 30, 3% OV-1; 3% QF-1; 1,5% OV 101,
i dexil 300.
Bazat pe o comparaie a metodelor menionate mai sus [90, 120, 122], rezultatele
optime au fost
realizat cu ajutorul unui OV 101 coloan de lungime min 50 i o temperatur de
120 capilar-170C
[118]. O OV-1 coloana a dat un bun de separare n mod similar pentru oxizi de
bisabolol A, B, C, bisabolol,
i bisabolone oxid. Separarea de izomeric cis-i trans-en-yne-dicycloethers a fost
realizate de ctre urmtoarele condiii prezentate n tabelul 10.5, utiliznd bisabolol
ca intern
standard. timpul de retenie sa a fost stabilit la 100 i sunt folosite ca referin.
n afar de calcule cu ajutorul unui integrator HP (18850 A-GC-terminal),
coninutul de absolut
de principii active n 100 g de material vegetal poate fi determinat de standard
intern
Metoda de diluare [16]. Mai multe experimente au fost efectuate utiliznd
hexadecanol ca intern
standard [31] Schilcher guaiazulena a dovedit c are avantaje peste hexadecanol n
mai multe
respect [121, 122].
TABEL 10.5
GC timpii de retenie a elementelor constitutive de musetel
Compus
Timpul de retenie / sec
Relativ timpul de retenie
Hexan
225
14
Farnesene
1047
66
Spathulenol
1340
85
Oxid de bisabolol B
1580
100
Bisabolon
1647
104
Bisabolol
1676
106
Matricin / chamazulene
1827
116
Oxid de bisabolol A
1936
123
Guaiazulena
2076
131
cis-en-yne-dicycloether
2546
161
trans-en-yne-dicycloether
2575
163
HP 5830 cromatograf. Coloane: OVI sau OV 101, 50 m. Temperatura program:
120 - 170 C, 5 C / min. C. Temperatura de injecie.: 225 C. Temperatura FID.:
250 Debitul de gaz: Heliu
la 1,2 ml / min (120 C) sau 1,9 ml / min (30 C). Hidrogen la 1,1 bar i oxigen la
1.7 bar. Atenuarea: 4 (pana la 7). de alimentare cu hrtie: 1,5 cm / min. Integrarea
cu HP
Integrator 18850 A.

Pagina 11
10.2.7 H
EADSPACE
G
AS
C
HROMATOGRAPHY
gaz cromatografie Headspace (HSGC) este o metod elegant pentru analiza
componentelor
de ulei esential cu mare precizie, a oferit un "head space" de extracie multiplu
(MHE) este per-
format. MHE previne matricea din interfereze cu analiza. Hiltunen et al. au
demonstrat
HSGC c este potrivit pentru analiza de ulei esential si active individuale principii
volatile
de musetel [39, 40, 41]. Folosind un DANI-HSS 3850 Automatic Head Space
Sampler, foarte precise
Rezultatele au fost obinute. Analizate au fost separate printr-un OV-1-coloan cu
variaie de
Temperatura de la 140 la 200C.
Stuppner i Bauer aplicat aceast metod pentru determinarea cantitativ a musetel
componente pregtire n ap i etanol, folosind un SF 52 coloan i o gam de
temperatur
de la 130-240C [144, 145]. Rezultatele au fost influenate de timp condiionat i
temperatur,
dar mai ales depinde de coninutul de etanol din eantion. la 5% etanol diluare n
ap
garantat rezultate acceptabile. n comparaie cu convenionale GC, HSGC a dat
valori aproape identice
pentru (-) - (|ioo|oo i derivatele sale, n timp ce valorile de en-yne-
dicycloethers au fost destul de redus [145].
10.2.8 H
IGH
-P
RESSURE
L
IQUID
C
HROMATOGRAPHY
Dezvoltarea unui protocol de separare cu ajutorul unui C Bondapak
18
coloana de la isocratic
i condiiile de gradient (amestecuri de metanol-ap) nu au avantaje n comparaie
cu publicarea
i metode CG TLC [122]. Alte investigatii constatat, de asemenea HPLC un mod
mai puin rezonabil "Strategia n
Cromatografie "[141] pentru cuantificarea componentelor din uleiul esenial. Cu
toate acestea,
HPLC este adecvat n special pentru separarea de compui izomerici. Azulenelor
separate foarte bine
pe-Chrosorb RP Li 8 coloan (faza invers), impregnate cu metanol-ap. Aceast
metod permite
Determinarea puritii de azulenelor izolate, precum i separarea de azulenelor
izomeric
(Figura 10.2) [121].
Figura 10.2 separare HPLC a azulenelor. Coloana: RP 8 alineatul (7 m); faz
mobil: metanol / ap 9 / 1;
lungime de und de detectare: 254 nm.
10 9
8
7
6
5
4
3
2
1
0
Guajazulen
Chamazulen
Matricin
Min.
TF4015_C010.fm pagina 231 vineri 2005-04-opt 08:25

Pagina 12
Pentru cuantificarea matricin n flori de musetel, Schmidt et al. [124, 126]
recomand
direct analiza HPLC a unui clorur de metilen extractul folosind Nucleosil 100-5
C
18
coloan i
un gradient de acetonitril / metanol / ap (12:35:53) ca solvent i metanol ca B cu
solvent
detectare de UV (244 nm).
Izomeric en-yne-dicycloethers pot fi separate pe o ODS SIL-x coloan (faza
invers), cu
% Acid acetic 2 n acetonitril (45:55, V / V) (Figura 10.3). n aceste condiii, nici
artefacte
nici pierderile sunt de ateptat [121].
Rezultatele bune de separare n mod similar au fost raportate de ctre Schulz
folosind ap / metanol (70:30, V / V)
[128]. Sumele constatate cu ocazia analizei HPLC sunt de aproximativ 16-39% mai
mare dect n GC. Pentru canti-
ficarea spiroethers, HPLC este cel mai bun, n timp ce analiza prin CSS este
complicat i consumatoare de timp.
Chamazulene acid carboxilic cantitativ a fost determinat n serul uman de ctre un
validat
testul HPLC. EDTA plasm a fost acidifiat cu acid fosforic, extras cu eter de
diizopropil,
centrifugated, evaporat, iar reziduul dizolvat n acetonitril. Separarea a fost realizat
pe o
RP-18 coloan, impregnate cu acetonitril-tampon pH 3 (4:6) i detecie UV (286
nm) [164].
10.2.9 E
NANTIOSELECTIVE
HPLC
Dup mai multe ncercri mai puin satisfctoare [12, 15, 34], Gnther et
al. stabilit un enantioselectiv
Metoda HPLC pentru a separa cele patru stereoizomeri
de (|ioo|oo [35]. posibile modificri ale
mueel ulei autentic pot fi identificate prin cuantificarea izomeri. (-) -
|ioo|oo are
efect mai puternic antiinflamatoare a tuturor stereoizomeri [51]. Este componenta
principal i indic
calitate de ulei de musetel. bisabolol sintetic (cotc un amestec de patru
izomeri. Sale antiinflamatoare
Activitatea este, prin urmare, mai slab [51] (a se vedea, de asemenea, Capitolul
4 asupra componentelor active). Cu toate acestea,
compus sintetic publicitate s fie "identic cu cel natural" este adesea folosit ca un
substitut sau
aditiv pentru ulei de musetel natural. Separarea de preparare a
izomerii (|ioo|oo a
fost realizate tribenzoylcellulose folosind ca faz staionar (coloana:
Superperformance, 10 mm
150 mm, 10-20 m). Eluent a fost un amestec de etanol / izopropanol / ap
(400:300:400, V / V / V;
debit 0,4 ml / min). Izomerii au fost detectate de un detector de UV (de tip "Soma
S-3702," 208 nm),
FIGURA 10.3 separare HPLC a izomerici en-yne-dicycloethers. fazei mobile;
ODS sol-x:: asul-Coloana
tonitrile / acid acetic 45/55% 2.
trans
cis
t (min)
1
2
3
4
5

Pagina 13
urmat de un polarimetru (546 nm). Authentic-bisabolol izomerii ( obinute de la
alte naturale
surse [34, 35] au fost utilizate ca substane de referin (a se vedea figura 10.4).
Acesta a fost primul raport cu privire la separarea celor patru izomeri fara izolare
de lung durat i
curat-up proceduri. Probele pot fi n continuare analizate folosind spectrometria de
mas izotopice (SM)
i
2
H-RMN-spectroscopie [12, 15]. Coloane cu un diametru mai mare permite o
semipreparative
analiz a izomerilor folosind presiune joas (LP) condiiile de LC. Puritatea de
izomeri obinut este
bun (exces enantiomeric aproximativ 95-98%) [34, 35].
10.2.10 D
ROPLET
C
OUNTERCURRENT
C
HROMATOGRAPHY
Droplet cromatografie n contracurent (DCCC) a fost aplicat cu succes de ctre
Becker et al. pentru
separate, ct i apolari compui polari [5]. Solventul a fost un amestec de hexan,
acetat de etil,
nitrometan, i metanol (9:2:2:3). Cu toate acestea, GC a dat rezultate mai bune n
ceea ce privete cromatografic
separare grafic comparativ cu DCCC.
10.2.11 HPLC / MS
I
GC / MS
HPLC i GC combinate cu spectrometrie de mas permite determinarea imediat a
componentelor
n extract de mueel [16, 128]. fragmentarea Caracteristic compui pot fi observate
ionizare folosind diferite metode: electrospray ionizare (ESI) Presiune
orAtmospheric chimice
Ionizare (APCI). Datele statele membre pot fi comparate cu datele de substane de
referin n experimente
sau literatura de specialitate (a se vedea Figura 10.5 ).
LC / MS analiz ofer rezultate rapide n ceea ce privete calitatea unui ulei de
musetel pur [16].
10.2.12 R
EVIEW A
O
NALYTICAL
P
OSSIBILITIES
de debit diagram Schilcher (figura 4.26, pagina 89 n Referin 121) prezinta
detaliat n continuare
n ceea ce privete metodele de analiz chimic a ulei de musetel.
Figura 10.4 separarea analitic a celor patru-bisabolol izomerii ( pe
tribenzoylcellulose: 1a = (-) - ( -
bisabolol; 1d = (+)cti(|ioo|oo; 1c = ()cti(|ioo|oo; 1b = (+) -
(|ioo|oo. absorbana UV la 208
nm (linie completa), rotaia optic la 546 nm (linia punctat).
Absorbana
Opt. Rotaie
0
-
0
30
60
90
120
150
180
210
240
Timpul de retenie (min)
1a
1d
1b
1c
+

Pagina 14
10.3 analiza chimic a flavonoide
10.3.1 D
ETERMINATION A
F
LAVONOIDS
Substane care interfereaz cu analiza de flavonoide (de exemplu, carotenoidelor)
sunt de obicei eliminate prin
extracie. Trebuie s fie luat n considerare faptul c majoritatea flavonoide apolari
va fi, de asemenea, eliminate
n aceast etap, de exemplu, prin extracie cu tetraclorur de carbon [106]. n plus,
de extracie la cald
Rezultatele ap n 30-45% mai mici valori pentru flavonoizi comparativ cu
extracie metanol. Prin urmare, este
nu este posibil pentru a evita co-extracia i legate de substane clorofila prin
utilizarea apei.
Determinarea fotometric de flavonoide are multe avantaje, cu toate c valorile
absolute
sunt de fapt de aproximativ 20-30% mai mare. Alte metode dup cum a sugerat
Reichling et al. nu au artat nici
mbuntire [103]. Coninutul absolut de flavonoide variat 1,0 i 2,5% ntre ntr-
un studiu
de 102 plante disponibile comercial i determinarea n conformitate cu Referine
106 i 17. Doisprezece
mostre de material de origine diferite cultivate de Schilcher a artat valori ntre 0,3
i
2.96% [121, 122].
10.3.2 P
APER
C
HROMATOGRAPHY
(PC)
I
T
HIN
-L
Ayer
C
HROMATOGRAPHY
Flavonoidele au i efecte neurotrop musculotropic pe spasmolysis. Prin urmare,
exist o farma-
interes macological n coninutul de flavonoide. Este cunoscut faptul c biosintez
flavonoide este influenat
de factorii de mediu. Identificarea i cuantificarea flavonoide elucideaz chimio-
taxonomie de material vegetal.
10.3.2.1 hrtie cromatografic
cromatografie pe hrtie ofer rezultate rapide folosind izopropanol / conc. acid
formic / ap (02:05:05,
V / V) ca solvent [46]. filtru de cromatografie ciclice pe filtre rund Ederol [111]
este, de asemenea, posibil.
FIGURA 10.5 Thermospray LC-MS cromatograma (un singur modul de ion m / z
= 201) a unei apoase / etanolic
floare extract de mueel (concentrat) i spectrele de mas a dou vrfuri
detectate. 1 = cis en-yne-eter, 2
Trans = en-yne-eter.
100
90
80
70
60
50
40
30
20
10
0
150
160
170
180
190
200
202
201
210
0
5 min.
0
2000
4000
6865
M / z 201
1
2

Pagina 15
10.3.2.2 cromatografie n strat subire
Dezvoltarea unor metode TLC a fost de succes pentru separarea de aproximativ 30
de flavonoizi,
printre care cele cinci aglyca principale, apigenina, luteolina, quercetin, patuletin, i
isorhamnetin, 20
glicozide flavonoide i aa-numitele "lipofile" flavone methoxylated [71]. Aceasta
a fost urmat de
determinarea densitometric unor constitueni principale, cum ar fi apigenina-7-
glucozid, quercetin-
7-glucoside, quercetin-3-galactoside, and patuletin-7-glucoside.
Silica gel 60 F
254
was used in combination with ethylacetate/formic acid conc./water (10:2:3)
[38], ethylacetate /formic acid/water (100:10:15) [71] or ethylacetate/formic
acid/glacial acetic
acid/water (100:11:11:26) [158] as eluent.
Aglyca were best separated on polyamide plates (Polygram DC11, MN) and a
solvent system of
methanol/methylethyl ketone/acetyl acetone (10 + 5 +1) [19] or
toluene/methylethyl ketone/methanol
(60 + 24 + 14) [103]. For the separation of aglyca on silica gel,
toluene/ethylformiate/formic acid (50
+ 40 + 10) is suitable [158]. Nineteen flavonoids were analyzed by this method
[70], not including the
methylated flavonoids (derivatives of the 6-methoxyquercetin and the 6,7-
dimethoxykaempferol) [147].
The main components mentioned above can be quantified using a Shimadzu CS
900 scanner. The TLC
plates were analyzed at two wavelengths (350 nm and reference wavelength of 710
or 460 nm).
Pretreatment of the TLC plate was not necessary. The peak height of the signals
was compared to
standard substances. For the TLC separation, the solvent system of ethyl
acetate/formic acid/water (10
+ 2 + 3) can be used.
For qualitative analysis the TLC plate is sprayed with Natural Products (NP)-
Polyethylene
Glycol (PEG) reagent. NEU-reagent consists of 1% methanolic diphenylboric acid-
-ethylamino
ester (NP) followed by 5% of ethanolic polyethylene glycol-4000 (PEG). The spots
were detected
by UV at 356 nm (see Figure 4.27 in Reference 121).
10.3.2.3 Two-Dimensional Thin-Layer Reaction Chromatography
Flavonoid glycosides as precursors of flavonoid aglyca can be detected using the
two-dimensional thin-
layer reaction chromatography [37, 67]. After the first development of the TLC
plate (sorbens: Silica
gel 60 F
254
), the sugars were hydrolyzed by hydrochloric acid applied in a microwave vapor-
blast
proces. Turning the TLC plate by 90 a second development was carried out
(solvent system: ethyl
acetate/formic acid/water 8 + 1 + 1 for both developments). The spots were
detected by NP/PEG-
reagent. Tschirsch and Hlzl adapted this method to acylated flavonoid glycosides,
eg, acylated
apigenin-7-glucoside-derivatives from chamomile [146] ( Figure 10.6 ) . The acyl
groups were removed
by saponification, liberating the flavonoid glycoside. In this case concentrated
ammonia was used after
the first development ( Figure 10.7 ). The duration and temperature of treatment
varied from 1 min at
room temperature to 1 hour at 70C depending on the stability of the acyl group
(sorbens: silica gel
60 F
254
; solvent system: ethylacetate/formic acid water 100 + 10 + 5 for both
developments, detection
by NP/PEG-reagent). The separation of apigenin-7- -D-(6"- O -acetyl)- and (4"-
O -acetyl)glucoside was
possible [146]. The method is particularly useful when no authentic reference
substances are available.
10.3.3 H
IGH
-P
RESSURE
L
IQUID
C
HROMATOGRAPHY
The first HPLC separation of quercetin, quercetin-7-glucoside, apigenin-7-
glucoside, rutin, herni-
arin, umbelliferone, and two unidentified phenyl carboxylic acids was done by
Schilcher [117,
118]. All compounds were separated on an RP 8 column running a gradient of
methanol-water
(1580%). The method was further developed and perfected for the qualitative and
quantitative
determination of chamomile flavonoids by Redaelli et al. [99, 100] in 1981 and by
Dlle et al. n
1985. A comparison of three methods is shown in Table 10.6 .
The method Dlle published allows reproducible determinations of the chamomile
flavonoids
[18]. Method no. 3 uses the diode-array technique for detection, giving reliable
results. Mai multe
later publications used this detection method [14, 82, 124, 125, 126, 128, 146].
TF4015_C010.fm Page 235 Friday, April 8, 2005 8:25 AM

Pagina 16
FIGURE 10.6 Two-dimensional thin-layer chromatogram without reaction of
ammonia. 1 = Apigenin-7-
glucosid; 2 = derivative 1; 3 = derivative 2; 4 = Apigenin-7- -D-(6 3 - O -
acetyl)glucoside; 5 = derivative 3; 6
= Apigenin-7- -D-(4 3 - O -acetyl)glucoside; 7 = Apigenin-7-glucosid control.
FIGURE 10.7 Two-dimensional thin-layer chromatogram after reaction with
ammonia for 1 min. 1 = Api-
genin-7-glucosid; 2 = derivative 1; 3 = derivative 2; 4 = Apigenin-7- -D-(6 3 -
O -acetyl)glucoside; 5 = derivative
3; 6 = Apigenin-7- -D-(4 3 - O -acetyl)glucoside; 7 = Apigenin-7-glucosid
control.
Star
t 2
1, 2, 3, 4, 5, 6
F
ront 2
Start 1

Fata
7
6
5
4
3
2
1
Star
t 2
1, 3, 4, 5, 6
F
ront 2
Start 1

Fata
7
6
5
4
3
2
1

Pagina 17
Carle et al. successfully separated the acetylated isomers of the apigenin-7- O - -
glucoside using
thermospray liquid chromatography/mass spectrometry (TSP LC/MS) [13]. The
analysis was per-
formed on an HPLC chromatograph HP 1090 L interfaced with an HP 5988A
thermospray MS.
The mass range scanned was m/z 150800 using positive and negative ionization
mode.
10.4 CHEMICAL ANALYSIS OF THE COUMARINS
The solvent system that was applied for the components in essential oil can be used
to separate
umbelliferone and herniarin in the dichloromethane extract on silica gel
60/Hf 254. Table
10.4 summarizes possible mobile phase systems as well as the RF values of the two
coumarins.
Both compounds show a soft blue fluorescence in short-wave UV light. Under
long-wave UV,
umbelliferone has a very strong, light-blue fluorescence and herniarin, a darker
blue one. Cei puternici
fluorescence of both compounds can be used for the direct quantitative
determination. It is best to
use HPTLC plates and a mobile phase system of ether/toluene (1:1, V/V) saturated
with 10% acetic
acid [110]. The compounds can be determined in fluorescent light at 365 nm at a
wavelength of
460 nm [147].
HPLC with UV/Vis detection rapidly and reliably allows the separation and
identification of
the coumarins in purified aqueous chamomile extracts [126, 128, 129]. Good
results were obtained
on an HP 1090 liquid chromatograph with microbore column. The mobile phase
was a gradient of
TABLE 10.6
Comparison of Three HPLC Separation Protocols (after Dlle et al. [18])
Metoda
1
o
2
3
Mobile Phase
Eluent A
2000 ml water
40 ml glacial acetic acid
1800 ml KH
2
PO
4
(0.005 mol/l)
175 ml methanol
2000 ml KH
2
PO
4
(0.005 mol/l)
14 ml dil. phosphoric acid
(pH approx. 2.6)
110 ml acetonitrile
16 ml dil. phosphoric acid
(pH approx. 2.55)
Eluent B
acetonitrile
1750 ml methanol
300 ml acetonitrile
1200 ml acetonitrile
600 ml methanol
Coloan
Coloan
material
Dimensiunea particulelor
Column size
Productor
RP 18
5 m
250 4.6 ID (steel)
Perkin Elmer
RP 8
10 m
250 4.0 ID (steel)
Merck
RP 18
5 m
125 4.0 ID (steel)
Merck
Parametrii
Injected
Volumul
15 l
15 l
15 l
Temperatur
37
35C
37
Debitul
1.0 ml/min
0.75 ml/min
1.0 ml/min
Sensibilitate
21 l0
-4
AU/cm
16 l0
-4
AU/cm
64 10
-4
AU/cm
Lungime de und
335 nm
350 nm
335 nm
Gradient
2785% B within 28 min
o
Modified according to Redaelli et al. [97100].

Pagina 18
phosphoric acid (pH 2.8) in acetonitrile. The compounds were identified using an
HP 1040A HPLC
sistem de detectare. The diode-array technology enables the simultaneous
measurement of the absor-
bance in a range of 190 to 600 nm. An HP 1046 A programmable fluorescence
detector can also
be used [129] (see Figure 10.8).
For the isolation of both coumarins, sublimation is a suitable method [120].
10.5 CHEMICAL ANALYSIS OF THE CHAMOMILE MUCILAGE
The extraction of chamomile flowers with 96% ethanol precipitates large amounts
of (natural)
mucilage. The mucilage has to be demineralized in order to determine its viscosity
and identify
individual polysaccharide components. This can be done using amberlite IR-120,
an acidic cation
exchanger, and amberlite IR-45, a basic anion exchanger. Comparative hydrolytic
tests [120] showed
that hydrolysis with trifluoroacetic (TFE) acid (1 ml of 1% solution of chamomile
mucilage + 1
ml 4N TFE, boiled for 30 min) is the most suitable method. Table 10.7 lists several
solvent systems
for the TLC separation of monosaccharides and urone acids.
Good separations of the monosaccharides and urone acids are shown in Figures
4.28 and 4.29
in Reference 121, page 92.
A selection of spray reagents is summarized in Table 10.8 . On heating the plates,
color reactions
s apar. However, for densitometric quantification, only immersed plates yield
reproducible results.
Franz et al. [25, 26] extracted the polysaccharides with cold water for 7 hours,
using chamomile
flowers that were pre-extracted with petrol ether and methanol, followed by
precipitation with ethanol
(final ethanol concentration 80% G/G). For the fractionation of the polysaccharides,
both ion exchange
and gel permeation chromatography (GPC) are recommended. Ion exchange
chromatography is
performed on DEAE-Sephacel columns in phosphated form by successive elution
with water/phos-
phate buffer (0.25/0.5/1.0 M ) and water/NaOH (0.2 M ). The polysaccharide
fractions are detected via
an anthrone test, dialyzed (MWCO 3500 D), and lyophilized. The latter is
performed by medium
FIGURE 10.8 Reversed phase HPLC and UV/Vis spectra of an aqueous extract of
fresh Matricaria chamo-
milla. A = flowers, B = leaves (eluent: diluted phosphoric acid, pH = 2,8/-
acetonitrile; detection: 337 nm). 1
= chlorogenic acid, 2 = caffeic acid, 3 = umbelliferone, 4 = hydroxy-cinnamic acid
derivate, 5 = luteolin-7-
glucoside, 6 = apigenin-7-glucoside, 7 = herniarin, 8 = apigeninglucoside (not
specified), 9 = apigenin-7-(6 3 -
O -acetyl)-glucoside, 10 = apigenin, 11 = cis En-In-Ether, 12 = trans En-In-Ether.
140
120
100
80
60
40
20
0
MAU
25
5
10
15
20
1
2
3
4
5
6
7
8
9
10
11
12
O
B
Timp (min.)

Pagina 19
pressure-GPC on HiLOAD 16/50 Superdex 75 or 200 columns. The fractions of
polysaccharides were
detected with an RI detector (eg, ERC 7512 Benthron Scientific), dialyzed, and
lyophilized. The
determination of molecular weights was performed in the same GPC system [25].
10.5.1 Q
UANTITATIVE
S
PECTRAL
D
ENSITOMETRIC
D
ETERMINATION OF THE
M
ONOSACCHARIDES AND
U
RONE
O
CIDS
Spots of samples (concentration approx. 5 g) and the sugar test solutions were
applied on the
TLC plate (silica gel 60 plates Merck) by microcaps. The test substances were
dissolved in 10%
of isopropanol and diluted to the final concentrations of 0.25, 0.5, 0.75, 1.0, 1.25,
and 1.5 g/ l.
Galacturonic and glucuronic acid were used in concentrations ranging from 0.5 to
1.25 g/ l [121].
The TLC plates were developed twice over a maximum length of 10 cm each. The
solvent system
was ethyl acetate-isopropanol-glacial acetic acid-water (60 + 30 + 5 + 5). After
thorough drying of
the TLC plates, the spots were detected by immersion in Scheffer-Kickuth reagent
for 5 sec. The
plates were dried and heated for 8 min at a temperature of 120C, immediately
followed by in situ
de msurare. Instrument parameters were as follows:
Apparatus: Zeiss chromatogram spectral photometer KM3
Wavelength: 385 nm
Gap width: 0.5
F-stop: 6
Gap measuring head plate: 2.5
Table speed: 200 mm/min.
Recorder 120 mm/min.
Evaluation: F = hxb
h/2
(h = peak height, b
h/2
= peak width at half level) or by evaluation
of the peak height
10.6 ANALYSIS OF CHAMOMILE: SUMMARY
The gravimetric method gives exact quantitative determinations of the total
essential oil.
In case of problems with the exact quantitative determination of individual
constituents,
a 1-hour extraction with methylene chloride is recommended.
For the analysis of the individual components in the essential oil, both TLC (eg,
on silica
gel plates GF
254
, mobile phase methylene chloride-ethyl acetate 98 + 2) and GC (OV 101
capillary column of 50 m length) are suitable. Very exact values of the spiroethers
can be
obtained by HPLC ( Figure 10.3 ).
In the quantification of the total flavonoids, lipophilic flavonoids should also be
included.
For the analysis of individual flavonoids, TLC is suitable (see Figure 4.27 in
Reference
121, page 90). Quantification can be done with TLC scanners, applying the two-
wave-
length technique (eg, on a Shimadzu CS 900).
For the determination of coumarins, TLC or HPLC and UV/Vis detection are best
when
using the same extract as for the analysis of the essential oil. The coumarins are
prefer-
entially isolated by sublimation.
For the analysis of components of mucilage, hydrolysis with trifluoroacetic acid is
particularly suitable. The chromatographic separation of saccharides and urone
acids can
be performed equally well either by thin layer, ion exchange, or gel permeation
chro-
matography. Quantification works well after immersion of the TLC plates in
Scheffer-
Kickuth reagent (see Figure 4.28 in Reference 121, page 90).

Pagina 20
REFERINE
1. (1971) Pharmacopoea Helv. Edit. VI.
2. (1978) Ph. Eur. III , 269271.
3. Adachi, S. (1965) J. Chromatog. 17 , 295.
4. Baltes, W., Liesk, J., Domesle, A. (1973) Chem.
Mikrobiol. Tehnologie. Lebensm. 2 , 92.
TABLE 10.7
RF Values of Monosaccharides and Uronic Acids with Various Mobile Phases
on Commercial Silica Gel 60 TLC Plates (Merck)
Carbohydrate
RF value
1
2
3
4
5
6
7
8
9
10
11
Galactoz
0.16
0.45
0.07
0.43
0.45
0.5
0.44
0.22
0.21
0.18
0.16
Glucoz
0.21
0.52
0.1
0.49
0.57
0.51
o
0.24
0.24
0.24
0.23
Arabinozei
0.23
0.64
0.13
0.57
0.64
0.65
0.48
0.28
0.27
0.31
0.30
Fucose
o
0.72
0.17
0.61
0.74
0.74
o
o
o
0.43
0.,7
Xylose
0.31
0.76
0.18
0.3
0.76
0.76
0.56
0.36
0.36
0.46
0.44
Ramnoz
0.39
0.88
0.28
0.69
0.85
0.86
0.63
0.2
0.2
0.62
0.57
Galacturonic acid
0
0
0
0.21
0
0
0.29
0.1
0.5
0
0
Glucuronic acid
0
0
0
0.23
0
0
0.0
0.14
0
0
0
1 : n-Propanol-Ethylacetate-Water
(70:20:10)
(V/V)
[3]
2 : Acetone-Water
(9010)
(V/V)
[95]
3 : Ethylacetate-aq. 2-Propanol (65%)
(65:35)
(V/V)
[142]
4 : Acetonitrile-Water
(85:15)
(V/V)
[27]
5 : Acetone-1-Butanol-Water
(50:40:10)
(V/V)
[4]
6 : Acetone-1-Butanol-Acetic acid-Water
(50:40:10:10)
(V/V)
[28]
7 : 1-Butanol-Acetic acid-Water
(80:30:30)
(V/V)
[163]
8 : Ethylacetate-Methanol-Acetic acid-Water
(60:15:15:10)
(V/V)
[36]
9 : 2-Propanol-Ethylacetate-Water
(50:40:10)
(V/V)
[94]
10 : Ethylacetate-2-Propanol-Water
(60:30:10)
(V/V)
[28]
11 : Ethylacetate-2-Propanol-Acetic acid-Water
(60:30 5:5)
(V/V)
o
No separation of fucose and xylose and of glucose and arabinose.
TABLE 10.8
Color Reactions of Monosaccharides and Galacturonic Acid after Immersion
of the TLC
Plates and Development at 100C
Anisaldehyde-
Acid sulfuric
Anilin-
Diphenylamin
-Naphthol-
Acid sulfuric
Carbazol-
Acid sulfuric
Scheffer-
Kickuth
Reagent
Galactoz
Dark green
Albastru
Rou
Blue-grey
Galben
Glucoz
Albastru
Blue-grey
Rou
Blue-grey
Galben
Arabinozei
Lumina verde
Verde
Rou-nchis
Blue-green
Galben
Fucose
Verde
Verde
Rou
Blue-green
Galben
Xylose
Lumina verde
Grey-green
Rou-nchis
Blue-green
Galben
Ramnoz
Verde
Pale green
Orange
Greyish pink
Galben
Galacturonic acid
Maro
Red-brown
Red-brown
Blue-green
Galben

nstudiulrobusteiimetodelorcromatograficepestratsubiretrebuiestudiatinfluenapeca
reomanifesturmtoriifactori:
temperatura;
umiditatea;
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forma i mrimea spoturilor;
compoziia fazei mobile;
pH-ul;
tipul de strat subire utilizat;
volumul de probaplicat;
forma bacului;
condiiile de uscare a placilor.

PentrumetodeleHPLCtrebuiestudiatinfluenaurmtorilorfactoriasuprarobusteiimeto
dei:
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temperatura n coloan;
tipul de coloan cromatografic;
volumul injectat;
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pH;
lungimea de und de detecie

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