LI CM N

Bi lun vn ny c thc hin v hon thnh ti phng nguyn cu chuyn su v ung

th ti trng i Hc Khoa Hc T Nhin, phng Linh Trung, qun Th c, thnh ph H
Ch Minh vi s hng dn ca.
Li u tin, em xin by t lng knh trng v bit n su sc ti PGS. TS. Nguyn Th
Thanh Mai cng ThS. Nguyn Xun Hi giao ti v tn tnh hng dn em hon thnh
lun vn tt nghip. Em xin chn thnh cm n cc thy c gio ca chuyn ngnh ha dc
khoa ha hc tn tnh ch dy v hng dn trong sut qu trnh hc v lm lun vn.
Em xin chn thnh cm n ThS. L Hu Th, anh on Vn Nht Trng hng dn,
gip em trong sut qu trnh lm lun vn.
Em xin gi li cm n ti cc thy c gio trng i hc Khoa hc t nhin i hc
Quc gia thnh ph H Ch Minh c bit l cc thy c gio trong khoa Ha, nhng ng i
du dt, gip v truyn t kin thc cho em trong sut thi gian theo hc ti trng.
Con xin gi li cm n ti b m, ng b, gia nh l ch da vng chc cho con hon
thnh kha lun.
Cm n nhng ngi bn, c bit l tp th lp ha dc K12 quan tm, ng vin,
gip ti hon thnh kha lun ny.
Tp. H Ch Minh, ngy thng nm 2016
Nguyn Vn My

TNG QUAN ............................................................................................................. 3

1.

2.

3.

4.

TNG QUAN C NGI BN................................................................................3

1.1.

Tn gi, m t thc vt v phn b.....................................................................3

1.2.

Dc l......................................................................................................... 4

1.3.

Thnh phn ha hc........................................................................................ 4

1.4.

Thnh phn chnh......................................................................................... 16

1.5.

Hot tnh sinh hc......................................................................................... 16

PHNG PHP SC K LNG HIU NNG CAO (HPLC) 27.................................21

2.1.

Khi nim v sc k...................................................................................... 21

2.2.

L thuyt HPLC........................................................................................... 22

2.3.

Phn loi..................................................................................................... 22

2.4.

Mt s i lng c bn trong phng php sc k lng......................................23

2.5.

Nguyn l hot ng...................................................................................... 29

2.6.

C ch tch................................................................................................. 30

2.7.

Cu to my................................................................................................ 30

U D UV28................................................................................................... 35
3.1.

u d hp th UV....................................................................................... 35

3.2.

u d DAD28.............................................................................................. 39

NHNG NGHIN CU V C NGI BN BNG HPLC.......................................46

4.1.

5.

Nui cy m t bo c Ngi Bn in vitro............................................................46

THM NH PHNG PHP35..........................................................................57

5.1.

nh ngha.................................................................................................. 57

5.2.

Ch tiu thm nh........................................................................................ 57

THC NGHIM.......................................................................................................... 82
TI LIU THAM KHO.............................................................................................. 83

TNG QUAN
1. TNG QUAN C NGI BN
1.1.

Tn gi, m t thc vt v phn b

Cy Ngi bn c tn khoa hc l Boesenbergia pandurata Roxb. Schltr.. Ngoi ra, cy Ngi

bn cn c tn thng l C ngi, Bng nga trut, Phn loi khoa hc ca cy Ngi bn c
trnh by trong bng 1.1.[1]
Bng 1.1: Phn loi khoa hc ca cy Ngi bn

Tn khoa hc
Gii
Ngnh
Lp
Phn lp
B
H
Chi
Loi

Boesenbergia pandurata Roxb. Schltr.

Plantae (Thc vt)
Angioperms (Thc vt ht kn)
Monocots (Thc vt mt l mm)
Commelinids (Thi li)
Zingiberales (Gng)
Zingiberaceae (Gng)
Boesenbergia
pandurata

Cy Ngi bn l cy thn tho. C Ngi bn mc tng c di, kt chm. C cn non c mu

mu trng, c gi mu vng lt. Khi b i c Ngi bn, bn trong c phn li mu vng m
hn phn tht mu trng c.
L c phin, gn l i xng. L di khong 30-60 cm, rng t 7-8 cm, cung di khong 512 cm.
Cy c t hoa, thng pht trin nch l. Hoa c dng ng, di 2-4 cm, vnh ng 1,5 cm, tai
khong 1,5 cm. Hoa c mu hng, mi hoa c kch thc 2,5 cm, bao phn 5 mm (Hnh 1.1). [1]
Cy Ngi bn c trng nhiu mt s nc nhit i bao gm Myanmar, Indonesia,
Malaysia, Thi Lan, 2
Vit Nam, cy c trng nhiu cc tnh pha Nam, ch yu An Giang, Tr Vinh,
R con l v tr tng hp cc cht trao i bc 2, thn r l ni lu tr. iu ny ph hp
vi cc nghin cu gn y cho thy chuyn v ca cc cht chuyn ha t gc n khc cc b
phn ca cy (. Dehghan et al 2013; Koo et al 2013.).

(a)

1.2.

(b)
Hnh 1.1: Cy Ngi bn: (a) Cy; (b) Hoa; (c) C

(c)

Dc l

Vit Nam, c Ngi bn c mi v nng v c s dng ph bin trong cc gia v ch bin

thc n. Trong dn gian, c Ngi bn c dng tr bnh hen suyn, tiu chy, kh tiu, nga,
st, lot, kh ming, kh chu d dy, kit l, [1]
Thi Lan, cy Ngi bn c tn l kra-chai. C ti dng lm nguyn liu trong ch bin
mn n, ngoi ra n cn c dng iu tr bnh hen suyn, tiu chy, chng ung th, cc chng
y bng kh tiu, chng nm, dit nm, chng vi khun, chng sng ty, gim au, h st, 2
Malaysia, cy Ngi bn c tn l temu kunchi; chi Boesenbergia phong ph nht ti
Malaysia v c s dng cha bnh au d dy.[3]
Indonesia, cy Ngi bn c tn l kunci pepet. C ti c dng trong nu n v s
dng trong cc bi thuc iu tr cc bnh nh l lot v kh ming, vim da, tiu chy. Ngoi
ra, c Ngi bn cn l thnh phn chnh trong mt loi thuc b dnh cho ph n c tn gi a
phng l Jamu.[4]
Myanmar, c Ngi bn c bit n di tn gi l seik-phoo, v c s dng rng
ri trong cc phng thuc dn gian iu tr cc bnh tiu chy, bnh hen suyn, chng kh tiu,
nga, st, [5]

1.3.

Thnh phn ha hc
1.3.1.

Cc hp cht chalcone

R1
OCH3
OH

R1
Geranyl
H

R2
OH
OCH3

Tn hp cht3, 6, 7, 8, 9, 10, 11, 12, 13

Pinostrobin chalcone
Cardamonin

Helichrysetin9

Tn hp cht9

OH
OCH3

2,4,6-Trihydroxydihydrochalcone
Uvangoletin

R2
OH
OCH3

R3
H
OH

Tn hp cht10
2,4-Dihydroxy-3-geranyl-6-methoxychalcone
Flavokawain C

2,6-Dihydroxy-4-methoxydihydrochalcone12
R
H
OCH3

Tn hp cht14
2-Hydroxy-4,6-dimethoxychalcone
2-Hydroxy-4,4,6-trimethoxychalcone

Hnh . Cc hp cht chalcone phn lp c t c Ngi Bn

1.3.2.

R1
OCH3
OH
OH

R2
OH
OH
OCH3

Cc hp cht Prenylchalcone

Tn Cht2, 10, 12, 15, 16

(-)-Panduratin A
(-)-4-Hydroxypanduratin A
(-)-Isopanduratin A

R1
OH
OH
OCH3

R2
OH
OCH3
OH

R1
OCH3
OH
OH

R2
OH
OH
OCH3

Tn Cht12, 17, 16
(+)-Panduratin A
(+)-4-Hydroxypanduratin A

(+)-Isopanduratin A

Tn hp cht10, 12
(-)-2-Hydroxyisopanduratin A
(-)-Nicolaioidesin B

()-Isopanduratin A1

(+)-Panduratin D5

(+)-Panduratin E5

(+)-Panduratin F5

(-)-Panduratin G5

(+)-Panduratin B1 v B25

Isopanduratin A2 10, 12

(+)-Panduratin C9

(+)-6-Methoxypanduratin A10

Boesenbergin B10

Boesenbergin A6
7

R1
OCH3
OH

R2
OH

Tn hp cht12
(+)-Nicolaioidesin C

OCH3

((+)-Karachaizin A)
(+)-Krachaizin B

R1
OCH3
OH

R2
OH

Tn hp cht12
(-)-Nicolaioidesin C

OCH3

((-)-Karachaizin A)
(-)-Krachaizin B

Hnh . Cc hp cht prenylchalcone phn lp c t c Ngi Bn

1 Cc hp cht Flavanonoid

R
Tn hp cht6, 12
OCH3 Pinostrobin
OH
Pinocembrin

Sakuranetin2,12

R1
OH
OH
OCH3

R2
Geranyl
Geranyl
H

R3
OCH3
OH
OH

R4
H
H
OH

Tn hp cht 10, 12
(2S)-6-Geranylpinostrobin
6-Geranylpinocembrin
7,4-Dihydroxy-5-methoxyflavanone

(2R)-8-Geranylpinostrobin10, 12

(2S)-7,8-Dihydro-5-hydroxy-2-methyl-2-(4methyl-3-pentenyl)-8-phenyl-2H,6H-benzo[1,2b:5,4-b]dipyran-6-one 10, 12

R
OCH3
OH

Tn hp cht12, 18
Rotundaflavone Ia, Ib
Rotundaflavone IIa, IIb

R
OH
OCH3

Quercetin12, 19

Tn hp cht12, 18
5,7-Dihydroxy-8-geranylflavanone
7-methoxy-5-Hydroxy-8-geranylflavanone

Kaempferol12, 19

5,7-Dimethoxyflavanone18, 12

R1
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3

R2
OH
OH
OCH3
OCH3
OCH3
OH
OH
OCH3
OH

R3
H
H
H
H
OCH3
H
H
H
OCH3

R
H
OCH3

R4
H
OCH3
H
OCH3
OCH3
H
OCH3
H
OCH3

7,4'-Dihydroxy-5-methoxyflavanone12

Tn hp cht18
Tectochrysin
5-Hydroxy-7,4'-dimethoxyflavone
5,7-Dimethoxyflavone
5,7,4'-Trimethoxyflavone
5,7,3',4'-Tetramethoxyflavone
5-Hydroxy-3,7-dimethoxyflavone
5-Hydroxy-3,7,4'-trimethoxyflavone
3,5,7-Trimethoxyflavone
5-Hydroxy-3,7,3',4'-tetramethoxyflavone

Tn hp cht12, 14
3,5,7,4-Tetramethoxyflavone
3,5,7,3,4-Pentamethoxyflavone

Hesperidin19

10

Naringin19
Hnh . Cc hp cht flavaonoid phn lp c t c Ngi Bn

1.3.3.

Cc hp cht polyphenol khc

2,4-Dihydroxy-6-phenethylbenzoic acid methyl ester 12, 19

axit Chlorogenic19

Dihydro-5,6-dehydrokawain2, 19

axit Caffeic19

Geranyl 2,4-dihydroxy-6-

Geranyl-2,4-dihydroxy-6-phenylbenzoate 19
phenethylbenzoate10

11

Axit p-coumaric19

5,6-Dehydrokawain10, 12, 19

2,4-Dihydroxy-6-phenethyl-benzoic acid methyl ester 19

Hnh . Cc hp cht polyphenol khc phn lp c t c Ngi Bn

1.3.4.

Cc hp cht tinh du

20

Tricyclene :

Camphor

Linalool

Camphene

-Pinene

-Pinene

-Terpineol

-Phellandrene

-Terpinene

Terpinolene

Terpinen-4-ol

-Elemene

-Elemene

-Elemene

12

Sabinene

-Thujene

cis-Linalool oxide

3-Carene

(Z)--Ocimene

13

(E)--Ocimene

14

15

Hnh . Cc hp cht tinh du phn lp c t c Ngi Bn20,21

1.4.

Thnh phn chnh

1.5.

Hot tnh sinh hc

Cc hp cht phn lp t c Ngi bn c th hot tnh khc nhau nh gy c t

bo ung th: ung th v MCF-7, ung th rut LS174T, ung th tuyn ty ngi PANC1, ung th t bo mu ngi HL-60, ung th i trng HT-29; khng virus HIV-1;
16

khng oxy ha nh c ch gc t do NO, c ch qu trnh peroxyde ha cht bo no

chut, khng amine d vng gy ung th; khng vim tai chut.
Nm 2001, Gassinee Trakoontivakorn v cng s[7] tin hnh th hot tnh chng li
cc hp cht amine d vng gy t bin nh 3-amino-1,4-dimethyl-5H-pyrido[4,3b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) v 2-amino-1methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) trn 6 hp cht phn lp c t c
Ngi bn ti Nht Bn thu c kt qu th hin trong bng 1.2.
Bng 1.2: Kt qu th hot tnh c ch cc cht Trp-P-1, Trp-P-2 v PhIP

Hp cht
(0.1 mol)
Pinocembrin chalcone
Cardamonin
Pinocembrin
Pinostrobin
4-Hydroxypanduratin A
Panduratin A

Trp-P-1

Phn trm c ch
Trp-P-2

PhIP

(50 ng)
93
98
94
95
91
87

(20 ng)
83
97
88
86
88
89

(250 ng)
82
88
85
86
82
81

Nm 2002, Patoomratana Tuchinda v cng s[2] tin hnh th hot tnh chng
vim i vi 2 hp cht 4-hydroxypanduratin A v panduratin A phn lp c t c
Ngi bn ti Thi Lan trn TPA (12-O-tetradecanoylphorbol-13-acetate) vi cht i
chng dng l diclofenac gy ra ph n tai chut thu c kt qu nh bng 1.3.
Bng 1.3: Kt qu th hot tnh chng vim

Hp cht

dy ph n tai

Phn trm c ch

sau 10h (m)

82,5 5
57,6 6
94 4

sau 10h
54
75
73

4-Hydroxypanduratin A (200 g/tai)

Panduratin A (200 g/tai)
Diclofenac (600 g/tai)

Nm 2003, Supinya Tewtrakul v cng s [22] tin hnh th hot tnh khng enzyme
protease HIV-1 (HIV-1 PR) trn 4 hp cht phn lp c t c Ngi bn ti Thi Lan,
vi cht i chng dng l acetyl pepstatin, kt qu thu c nh bng 1.4.
Bng 1.4: Kt qu th hot tnh khng enzyme protease HIV-1

Hp cht
Pinostrobin

Phn trm c ch
22,52 0,56
17

IC50 (g mL-1)
> 100

Pinocembrin
Cardamonin
Alpinetin
Acetyl pepstatin

25,48 0,44
75,11 1,44
23,76 3,65
98,47 0,27

> 100
31,0
> 100
0,32

Nm 2005, Sariga Zaeoung v cng s[23] tin hnh th hot tnh c ch gc t do

DPPH (1,1-diphenyl-2-picrylhydrazyl), gy c t bo ung th v MCF-7, gy c t bo
ung th rut LS174T ca tinh du, cao methanol, cao nc ca c Ngi bn ti Thi Lan
thu c kt qu nh bng 1.5.
Bng 1.5: Kt qu th hot tnh khng oxy ha, gy c t bo MCF-7 v LS174T

Mu th
Tinh du
Cao methanol
Cao nc

Phn trm c ch ti nng 100 g mL-1

DPPH
MCF-7
LS174T
5,4 1,2
0,6 0,7
5,2 0,5
47,4 3,5
20,6 7,7
89,9 2,4
11,3 1,6
92,6 14,3
79,4 6,0

Nm 2006, Kazutoshi Shindo v cng s [15] tin hnh th hot tnh c ch

peroxide ha lipid trn no chut ca 6 hp cht phn lp c t c Ngi bn ti
Nht Bn, vi cht i chng dng l catechin, kt qu thu c nh bng 1.6.
Bng 1.6: Kt qu th hot tnh c ch peroxide ha lipid

Hp cht
Pinostrobin
Pinocembrin
Panduratin A
4-Hydroxypanduratin A
Pinostrobin chalcone
Cardamonin
Catechin

IC50 (M)
230
15
210
70
38
4,5
17

Nm 2006, Sarot Cheenpracha v cng s [9] tin hnh th hot tnh khng enzyme
protease HIV-1 (HIV-1 PR) t 7 hp cht phn lp c t c Ngi bn ti Thi Lan,
vi cht i chng dng l acetyl pepstatin, thu c kt qu nh bng 1.7.
Bng 1.7: Kt qu th hot tnh khng enzyme protease HIV-1 PR

Hp cht
Panduratin C
Panduratin A
4-Hydroxypanduratin A
Helichrysetin
2,4,6-Trihydroxyhydrochalcone
Uvangoletin
18

IC50 (M)
> 100 (43,1 %)
18,7 0,8
5,6 0,7
> 100 (14,1 %)
> 100 (7,5 %)
> 100 (2,7 %)

Cardamonin
Acetyl pepstatin

> 100 (47,6 %)

3,4 0,2

( ): % c ch ti nng 100 M
Nm 2006, Chandra Kirana v cng s [4] nghin cu v pht hin hp cht
panduratin A c phn lp t c Ngi bn ti Singapore c kh nng gy c t
bo ung th v MCF-7 (IC50 = 3,75 M) v t bo ung th i trng HT-29 (IC 50 =
6,56 M).
Nm 2007, Nwet Nwet Win v cng s [10] tin hnh th hot tnh gy c dng
t bo ung th tuyn ty ngi PANC-1 trn 24 hp cht phn lp c t c Ngi
bn ti Myanmar, vi cht i chng dng l arctigenin thu c kt qu nh bng
1.8.
Bng 1.8: Kt qu th hot tnh gy c t bo PANC-1

PC100

Hp cht

(M)

Nicolaioidesin B

2,5

Panduratin A
Isopanduratin A1
6-Geranylpinocembrin
Geranyl-2,4-dihydroxy-6-phenethylbenzoate

2,4-Dihydroxy-3-(1-geranyl)-6-methoxychalcone
2-Hydroxyisopanduratin A

16

Isopanduratin A2
4-Hydroxypanduratin A
7,8-Dihydro-5-hydroxy-2-methyl-2-(4-methyl-3pentenyl)-8-phenyl-2H,6H-benzo[1,2-b:5,4-b
]dipyran-6-one
Pinocembrin

64

6-Methoxypanduratin A
Boesenbergin A
Boesenbergin B
8-Geranylpinostrobin

128

6-Geranylpinostrobin
Flavokawain C

> 256

Cardamonin
19

Pinostrobin
Alpinetin
7,4-Dihydroxy-5-methoxyflavanone
Panduratin C
Tectochrysin
5,6-Dehydrokawain
Taxol
Arctigenin

> 256
1

Nm 2007, Mohd Aspollah Sukari v cng s [11] tin hnh th hot tnh gy c
dng gy ung th t bo mu ngi HL-60 trn cao v 5 hp cht phn lp c t c
Ngi bn ti Malaysia, thu c kt qu nh bng 1.9.
Bng 1.9: Kt qu th hot tnh gy c t bo HL-60

Cao / Hp cht
Hexane
Chloroform
Methanol
Boesenbergin A
Alpinetin
Cardamonin
Pinostrobin
Pinocembrin

IC50 (g/L)
8,5
5,8
> 30,0
5,8
11,0
23,2
> 30,0
> 30,0

Nm 2009, Supinya Tewtrakul v cng s[24] tin hnh th hot tnh c ch vic
sn sinh ra gc t do NO trn t bo RAW264.7 vi cht i chng dng l caffeic acid
phenethyl ester t cc hp cht phn lp c ca c Ngi bn ti Thi Lan kt qu thu
c nh bng 1.10.
Bng 1.10: Kt qu th hot tnh c ch vic sn sinh ra gc t do NO trn t bo RAW264.7

Hp cht
Panduratin A
4-Hydroxypanduratin A
Cardamonin
Helichrysetin
Panduratin C
Uvangoletin
2,4,6-Trihydroxyhydrochalcone
L-Nitroarginine (L-NA)
20

IC50 (M)
5,3
13,3
24,7
62,3
67,8
74,7
96,2
61,8

Caffeic acid phenethyl ester

1.5.1.

5,6

Hot cht chnh

2. PHNG PHP SC K LNG HIU NNG CAO (HPLC)25

2.1.

Khi nim v sc k

Sc k l nhng k thut tch v phn tch (xc nh) cc cht trong mt hn hp mu

da theo nhng tnh cht ha hc, vt l v ha l ca cc cht trong nhng iu kin nht nh.
K thut sc k c hai loi da theo trng thi ca cht mu v pha ng khi tin hnh tch sc
k: K thut phn tch sc k kh (GC), pha ng l cht kh; k thut phn tch sc k lng
(LC), pha ng l cht lng.
Trong sc k lng chia thnh hai nhm: Sc k lng p sut thng, LC (sc k c in);
sc k lng hiu nng cao (High Performance Liquid Chromatography, HPLC), lm vic vi p
sut t 150-250 bar.
T nm 1999 c sc k lng siu p (Ultra High Performance

Liquid

Chromatography, UHPLC), lm vic vi p sut cao t 900-1300 bar.

So vi k thut sc k lng p sut thng, k thut HPLC hin nay ang c pht
trin rt nhanh v c ng dng rt rng ri trong nhiu lnh vc khc nhau trong nghin cu
khoa hc, sn xut cng nghip, mi trng,K thut tch sc k HPLC c nhy cao, hiu
sut tch cao v tn t thi gian cng nh tn t mu, chim n 70% tng s cc cng trnh
nghin cu v ng dng v sc k. K thut tch sc k HPLC gm hai nhm: Sc k lp mng
hiu nng cao (HPTLC) v sc k ct lng hiu nng cao hay cn gi l sc k lng hiu nng
cao (HPLC)

2.2.

L thuyt HPLC

Phng php sc k lng hiu nng cao (HPLC) ra i nm 1967-1968 trn c s pht
trin v ci tin t phng php sc k ct c in. HPLC l mt phng php chia tch trong
pha ng l cht lng v pha tnh cha trong ct l cht rn c phn chia di dng tiu
phn hoc mt cht lng ph ln mt cht mang rn, hay mt cht mang c bin bng lin
21

kt ha hc vi cc nhm chc hu c. HPLC s dng cao p lc y dung mi qua ct cha

cc ht rt mn cho phn gii tch cao.
Phng php ny ngy cng c s dng rng ri v ph bin v nhiu l do: c nhy
cao, kh nng nh lng tt, thch hp tch cc hp cht kh bay hi hoc d phn hy nhit, c
phm vi ng dng tri rng trong nhiu lnh vc t nghin cu khoa hc trong cc phng th
nghim n cng nghip v mt s lnh vc khc.

2.3.

Phn loi

Da vo s khc nhau v c ch tch chit s dng trong HPLC, ngi ta chia HPLC thnh 4
loi:

Sc k hp ph hay sc k lng rn (adsorption/liquid chromatography).

Sc k phn b (partition chromatography).

Sc k ion (ion chromatography).

Sc k ry phn t (size exclusion/gel permeation chromatography).

Trong , sc k phn b (SKPB) c ng dng nhiu nht v c th phn tch c nhng

hp cht t khng phn cc n nhng hp cht rt phn cc, hp cht ion c khi lng phn
t khng qu ln (<3000). SKPB c chia thnh hai loi da trn phn cc tng i gia
pha tnh v pha ng: sc k pha thng SKPT (normal phase chromatography) v sc k pha
o SKP (reversed phase chromatography). Pha tnh trong HPLC l mt yu t quan trng
quyt nh s tch sc k ca mt hn hp cht mu phn tch.
Trong SKPT, pha tnh s dng c phn cc cao hn pha ng. Pha tnh loi ny s c i
lc vi cc hp cht phn cc. SKPT dng tch v phn tch cc hp cht c phn cc cao
vi phn t lng khng ln lm.
Cc loi ct pha tnh trong pha thng: Ct Silica gel; ct Silicagel bin tnh: Ct Cyano
(dng a mc ch), ct Amino (phn tch ng), ct Diol (phn tch protein). Dung mi pha
ng s dng cho sc k lng pha thun ch yu khng phn cc (dung mi chnh):
Hydrocarbons (Pentane, Hexane, Heptane, Octane), Aromatic Hydrocarbons (Benzene, Toluene,
22

Xylene), Methylene chloride, Chloroform, Carbon tetrachloride. Ngoi ra cn c dung mi phn

cc hoc hi phn cc (dung mi ph): Methyl-t-butyl ether (MTBE), Diethyl ether,
Tetrahydrofuran (THF), Dioxane, Pyridine, Ethyl acetate, Acetonitrile, Acetone, 2-propaol,
ethanol, methanol
Dung mi chnh c s dng chnh lm pha ng, dung mi ph thng c thm vo
vi t l nht nh thay i thi gian lu. Cc dung mi thng khng hp thu trong vng UV
d dng cho vic xc nh.
Trong SKP, pha tnh t phn cc hn pha ng. Phng php ny dng phn tch cc
hp cht t khng phn cc n phn cc. Hu ht cc hp cht hu c c mch carbon di (t
phn cc) rt thch hp cho phn tch bng SKP. Dung mi s dng trong SKP l cc dung
mi phn cc, trong nc ng vai tr quan trng m li r tin. Do , SKP c ng
dng nhiu v ph bin hn SKPT.
Cc lai ct pha o: Ct C18 (ODS), ct C8 (octyl), ct C4 (butyl), ct Phenyl, ct
TMS. Dung mi thng s dng nht cho sc k pha o l dung mi phn cc nh: Methanol
(MeOH), acetonitrile (ACN) hay THF v nc, trong nc ng vai tr quan trng m li r
tin, do sc k lng pha o c s dng nhiu nht. Khi s dng dung dch m, nng
m v pH l nhng thng s rt quan trng cn lu .Vic ti u ha t l ca dung dch m v
dung mi hu c cng rt quan trng.Vi cng mt lai vt liu nhi ct, ty theo kch c ht
nhi ct, hng ch to ct thnh phn pha ng cng c th phi thay i cho ph hp.

2.4.

Mt s i lng c bn trong phng php sc k lng

2.4.1.

Thi gian lu (tR, retention times )

Thi gian lu l thi gian tnh t lc np mu vo ct tch sc k cho n cht tan c

ra gii ra khi ct im c nng cc i, c trng cho mi cht phn tch.
tR = tO + tR
Trong , tO : Thi gian khng lu gi (thi gian cht tan nm trong pha ng cng l thi gian
pha ng i t u ct n cui ct v cn gi l thi gian lu cht)
tR : Thi gian lu gi thc ca cht tan X trong ct sc k ( pha tnh)

23

Hnh 7. Thi gian lu ca cu t phn tch

Thi gian lu tR ca mt cht tan trong ct sc k ca qu trnh sc k ph thuc vo nhiu yu
t nh: Bn cht pha tnh, kch thc, xp, cu trc xp; Bn cht, thnh phn, pH, tc pha ng;
Cu to v bn cht ca phn t cht tan X Trong , pH ca pha ng, nng cht to phc nh
hng r nt n cn bng ng trong qu trnh sc k cc cht ion v cp ion.
Gi tr thi gian lu tR c ngha rt quan trng trong thc t ca k thut sc k. N cho bit
cc cht tan X trong hn hp mu c ra gii nh th no trong cc iu kin th nghim v h pha
chn, ng thi dng cho nh tnh.

2.4.2.

H s phn b (K, partition coefficience), h s dung

lng (k, capacity factor)

2.4.2.1 H s phn b (K, partition coefficience)
Qu trnh tch sc k ca cht tan X da trn c s s phn b ca cht tan gia pha
ng (MP) v pha tnh (SP) xy ra lin tc trong ct sc k. S phn b ny c c trng bi
mt i lng gi l h s phn b K ca cht X, l t s nng ca cht tan X trong pha tnh
v pha ng, c tnh theo cng thc:
K

C X ( SP )
C X ( MP )

Vi CX(SP) : nng cu t trong pha tnh.

CX(MP) : nng cu t trong pha ng.

H s phn b K cho ta bit kh nng phn b ca cht tan X trong pha tnh v pha ng,
h s K ty thuc vo bn cht pha tnh, pha ng v cht phn tch.
24

2.4.2.2 H s dung lng (k, capacity factor)

H s dung lng l t s khi lng cht tan X phn b pha tnh v pha ng, v c
tnh bng cng thc:
k'

mX ( SP )
mX ( MP )

Nu gi t s VSP/VMP = q, l t s pha ca h sc k HPLC, ta c mi quan h gi h s

phn b v h s dung lng:
k ' K .q

V m(SP) = CSP.VSP, m(MP) = CMP.VMP, q = const, nn h s dung lng k nh l hm s ca

h s phn b K c dng hm s y = ax.
Trong phn tch HPLC h s dung lng k nm trong vng t 2-7 l thch hp. Nu h
s dung ln k nh hn 2 qu trnh tch khng tt, ln hn 7 th qu trnh ra gii s ko di,
chn pic s ging rng v nh hng n phn gii (s tch).
Gi tr k, k ph thuc vo bn cht v cu trc ca phn tch, pha tnh, thnh phn pha
ng, tc ra gii, nhit chy sc k, gi tr pH pha ng (cho h IE v IP-HPLC).

1 H s tch ()
H s tch hay cn gi l thi gian lu tng i ca hai cht A v B, c tnh bng t
l thi gian lu thc hoc l hng s dung lng ca hai cht A v B. H s tch ty thuc bn
cht v thnh phn dung mi pha ng, pH ca mi trng, bn cht ca pha tnh, nhit . Hai
cht tch c khi 1 v cng ln th s tch ca hai cht A v B cng r, = 1 khng c s
tch sc k gi A v B (hai pic sc k A v B trng nhau).

t ' A k ' A (t A tO )

t 'B k 'B
tO

k'

(t R tO )
tO

25

2.4.3.

S a l thuyt (N, theoretical plates ) v chiu cao a ca

ct sc k
S a l thuyt ca ct (N) trn 1 mt chiu di ct l i lng c trng cho ct sc k,
c coi nh l s lp cht tan c chiu cao l H. Chiu cao H ph thuc vo nhiu yu t:
-

ng knh ca ht pha tnh, hnh dng v kiu ht trn hay mnh;

iu kin ch to, xp, kch thc l xp ca ht pha tnh;
Bn cht, cu trc phn t ca cht tan (cht phn tch);
Loi, tc v thnh phn ca pha ng trong qu trnh sc k;
nht ca pha ng,
C c pH ca pha ng, nu l sc k ion v cp ion.

Vi mt h pha nht nh (SP v MP) v trong nhng iu kin sc k chn, th chiu

cao Hxi c nhng gi tr xc nh ng vi cc cht tan. Chiu cao Hxi ny c xc nh
theo cng thc:
H

L W 2
.( )
16 t R

Vi ct sc k c chiu di L, s a l thuyt c tnh theo cng thc:

N

t
L
16.( R ) 2
H
W

Trong , tR l thi gian lu ca cht tan v W l chiu rng y pic sc k ca cht tan.
nh gi ng kh nng v hiu ca ct sc k trong mi trng hp c th ngi ta
thng quan tm ti s a hiu dng Nef v chiu cao hiu dng Hef.
H ef

N ef

L W 2
.( )
16 t 'R

t'
L
16.( R )2
H ef
W

Vi tR l thi gian lu thc ca cht tan trong ct sc k

T cc cng thc trn ta thy Hef lun ln hn H v Nef lun nh hn N. ng thi Hef v
Nef l nhng hng s c trng cho nhng qu trnh tch sc k ca mt h pha vi hn hp mu
26

nht nh. Nu gi tr Nef l qu nh th qu trnh tch khng tt, hoc s tch khng hon ton.
Nef cng ln th hiu qu tch cng cao, Nef ca mt ct tch nm trong vng t 5000 8000 l
va , cho nhng cht khng qu phc tp . Nhng nu Nef l qu ln th cng khng cn thit.
V khi pic sc k ca cc cht tch xa nhau qu v vic ra gii tn nhiu pha ng.

2.4.4.

phn gii (R, resolution)

phn gii l i lng cho bit mc tch ca cc cht tan trong mt h pha.
phn gii c xc nh theo cng thc:
RAB

2.(t 'RA t 'RB )

(WA - WB )

Trong : tRA v tRB l thi gian lu thc ca A v B

WA v WB l b rng y pic sc k ca hai cht A v B

hai pic c th tch ra khi nhau th

WAB WA WB

. Nu R cng ln th hai pic ca

hai cht A v B cng xa nhau, dn n tn nhiu thi gian v dung mi ra gii. Do , gi tr

R ch cn va tch hon ton hai cht A v B ra khi nhau. Ngha l ch cn hai pic va

tch hn ra khi nhau, tc l

WAB WA WB

Hnh. Gin v s tch ca hai pic sc k A v B

27

(a) Ti thiu c th tch hai pic (R 0.75) (b) hai cht tch ra khi nhau (R 1)
(c) Hai cht tch xa nhau (R 1.5)

Trong thc nu R > WA +WB gim thi gian ra gii cn s dng qu trnh ra gii
gradient thnh phn pha ng (tuyn tnh hay tng bc) cht th hai c ra gii ra
nhanh hn.
Theo quy tc Rayley, ta c cng thc:
1 ( 1)
R .
.( N ef ) 2
4

hay

1
1 ( 1) k '
R .
.
.N 2
4 (k ' 1)

Cc cng thc ny cho php ta xc nh c cc h s phn gii ca h pha chn,

theo cc tham s k, N v trong mi trng hp c th. Mi quan h gia cc i lng R,
Nef v bng
Bng: Mi quan h gia N, R v
Gi tr

S a Nef ti thiu phi c

1.05
1.10
1.15
1.25
1.50

ng vi R
R =1.00, cn N
7.000
3.500
2.700
1950
1050

2.4.5.

R = 1.50, cn N
16.000
11.500
7.300
5.500
3.000

bt i xng (Asymmetry factor = tailing factors)

bt i xng ca pic l t s rng pic sc k na sau v na trc v tr ng vi

1/10 chiu cao cc i ca pic (Hmax). Nu gi rng ca na trc pic sc k l a v na sau
l b ti v tr 1/10 Hmax, ta c bt i xng Asys:
Asys = (b/a)
c phn gii tt, gi tr cho php ca bt i xng ca pic sc k A sys = 1 0.1. Khi
pic cn i th h s Asys = 1 l iu kin tt nht.

28

2.5.

Nguyn l hot ng

Mu sau khi c tim vo ct s c pha ng li ko qua ct. Da vo kh nng

tng tc khc nhau gia cc cht c trong nn mu vi pha tnh v pha ng m chng c
tch ra khi nhau v sau khi ra khi ct s c ghi nhn bi b d c th. Sc k lng c th
ghp vi nhiu loi u d khc nhau nh u d ph t ngoi kh kin UV-Vis, u d hunh
quang FLD, u d ch s khc x RID, u d in ha ECD, u d khi ph MS Hin nay
u d khi ph c ng dng rng ri trong phn tch vt v cc hp cht nhn danh chnh
xc.

2.6.

C ch tch

Qu trnh tch l qu trnh quan trng nht trong phng php sc k ni chung v sc k
lng ni ring. Hiu qu ca qu trnh ny ph thuc rt nhiu vo tng tc gia cc cht trong
pha tnh v pha ng. Mc ch chnh ca sc k l tch v nh tnh cc cht trong hn hp cht
phc tp. Tuy nhin trong thc t khng phi lc no vic tch cc cht cng xy ra han ho do
cc tng tc l rt phc tp v tng tc ca mi cht khi nm ring r v nm trong hn hp

29

li phn no khc nhau Do s tng tc khc nhau gia cc cht phn tch v vt liu nhi ct
m qu trnh tch xy ra.

2.7.

Cu to my

Thit b sc k lng hiu nng cao bao gm cc b phn chnh: bm, b phn tim mu,
ct sc k phn tch, u d, b phn iu khin v x l s liu.

2.7.1.

Bm cao p

Bm cao p bm pha ng (dung mi ra gii) qua ct sc k thc hin qu trnh tch

cc cht c trong mu tim vo ct sc k theo mt chng trnh ph hp. C hai kiu bm
chnh:
Bn nn trc tip pha ng t xy lanh (bm piston) vo h HPLC.
Bm nn y pha ng qua mt mng bm chu p lc cao (bm mng).
Trong hai loi bm ny, loi th bm hai c nhiu u im hn. c bit loi bm th
hai khng lm nhim bn pha ng v pha ng khng tip xc vi piston ca bm. C rt nhiu
loi bm da trn hai loi bm ny ra i v c ng dng ph bin trong k thut HPLC.
phc v cho mc ch tch phn tch, cc bm ny c th thay i p sut t vi chc bar n
1000 bar vi tc bm pha ng t 0.2 10 mL/pht, p sut np ct c th ln n 1500 bar.
Yu cu ca mt bm cao p dng cho k thut phn tch HPLC:
Khng lm bn pha ng gy nhiu cho qu trnh sc k
30

 Phi d dng iu chnh tc bm mong mun ca pha ng trong phm vi cho

php ca qu trnh chy sc k
bn cao
Vng thay i tc bm tng i rng c th phc v c cho nhiu qu
trnh sc k khc nhau (0.35 5 mL/pht), lp li tt.
Cc b phn chnh ca bm:

M t ng c bm
Bung bm v b o p lc
H van vo v van ra ca pha ng
Cc b kim tra v iu chnh p sut bm ht v y pha ng
B trn pha ng v n nhit
H in t iu khin v kim tra qu trnh lm vic ca bm

2.7.2.

B phn tim mu

B phn tim mu c dng tim chnh xc mt lng mu nht nh (hn hp phn

tch) vo ct sc k. C hai loi van bm mu, loi c vng mu v loi khng c vng mu.
Trong k thut HPLC, van bm 6 chiu c 1 vng mu l c s dng ph bin nht. Nguyn
tt cu to v lm vic ca loi van ny c m t trong hnh ?. Cc vng mu thng c th

31

tch l: 10, 20, 50 L cho mc phn tch v 1.000, 5.000, 10.000 L cho bn iu ch v iu
ch.
C hai cch tim mu: Tim mu bng tay s dng micro-xylanh, nh vng mu c th
tch xc nh; Tim mu t ng theo chng trnh. Khi lng mu phn tch nh hn th tch
vng mu m ta khng mun pha long mu th c th bm trc tip bng micro-xylanh.

Hnh. Cu to van bm mu 6 chiu trong HPLC

(a) Load- V tr np mu vo vng mu; (b) injection- V tr bm mu vo ct tch
2.7.3.

Ct sc k phn tch

Ct sc k l b phn chnh ca qu trnh sc k. Mi din bin ca s tch mt hn hp

cht phn tch u xy ra y v kt qu ca s tch tt hay khng ph thuc chnh vo ct.
Ct sc k trong HPLC c ch to bng thp c bit tr vi ha cht, chu c p
sut cao n vi trm b, c khi n 1.000 bar. Ngoi ct thp, cn c cc ct v ngoi bng thp
bc ng trong thy tinh hay polyme dng tch loi pha ng c mi trng axits cao v cht
to phc mnh m thp khng c kh nng chu ng c. Thng thng ct sc k c rng,
ng knh trong (id) t 2 - 4.5 mm, chiu di t 10 25 cm cho mc ch phn tch. ng
knh hay kch thc ct cng nh hng n kt qu tch sc k. Bn trong ct sc k cha pha
tnh thc hin tch cc cht trong mu phn tch. Cu to ct sc k nh trong hnh.

32

Hnh. Cu to ct sc k trong k thut HPLC

2.7.4.

u d

u d l thit b thu nhn v pht hin cc cht phn tch da theo mt tnh cht ha,
ha l hay vt l no ca cht phn tch hay dng hp cht ca n i ra t ct tch, v d nh
s hp thu quang, s pht quang, s thay i dng in, dn in, chit sut ca cht. Mt s
u d thng dng cho k thut HPLC.

u d ph hp thu quang phn t UV hay UV-Vis, hay PAD (DAD)

u d ph hunh quang nguyn t, phn t
u d ph pht x nguyn t, ICP-OES
u d ph hp thu nguyn t, AAS
u d khi ph
u d in ha o dng v cc ph
u d o in th (mV)
u d o dn in, hay in tr
u d o chit sut
u d o hiu ng nhit

Trong , u d hp thu quang phn t UV-Vis v PAD c s dng ph bin nht

trong k thut HPLC. Tip n l u d ph hunh quang nguyn t, phn t, u d khi ph,
u d in ha, u d o chit sut.
Mt s yu cu ca u d s dng trn k thut HPLC:
C tnh cht chn lc phn tch cao
C nhy cao vi cht cn phn tch trong hn hp mu. C th pht hin v nh
lng cc nng nh (ppm, ppb).
33

 Hot ng n nh v bn trong iu kin phn tch.

Vng tuyn tnh ca php o khng c qu nh. Cng rng cng tt cho vic pht
hin v nh lng c cc cht trong mt vng nng rng.
Khng qu phc tp v kh s dng
nhiu nh
Cc thng s nh gi cht lng ca mt u d:

nhiu: Phi nh v n nh theo thi gian

Gii hn pht hin (LOD): LOD vi cht phn tch cng nh cng tt (ppb-ppm)
Vng tuyn tnh: Cng rng cng tt 1- 104 ln.
dong pic do u d: Nh, khng c ln hn 10% tng dong pic chung

ca pic sc k, khng nh hng n cn i pic.

Hng s thi gian: Nh. Ngha l phi c s p tuyn nhanh v nhy vi mi s
thay i nh ca nng cht phn tch qua Bung o ca u d. Thi gian p
tuyn phi nh hn 0.1 giy.
lp li ca tn hiu o: Phi c lp li cao. o ti thiu 10 ln kt qu khng
c sai qu 10% i vi mt nng nm trong vng tuyn tnh.
Cu to c bn ca mt u d:
Bung (bung o) c th tch t 5 15 L.
H quang hc: Cung cp nh sng c bc sng thch hp cho phn tch
H in t o tn hiu: Thu nhn v khuch i tn hiu o n thit b ghi nhn v
x l hin th kt qu.
2.7.5.

B phn ghi nhn v x l hin th kt qu

Thng l mt my tnh ci t cc phn mn ghi nhn tn hiu t u d sau chuyn

i thnh tn hiu v hin th ln mng hnh my tnh.
Ngoi cc b phn chnh trn, ngy nay vi s pht trin ca khoa hc cng ngh nhiu
b phn c hon thin hn nh b chng trnh dung mi thay i thnh phn pha ng,
b np mu t ngp ng c nhiu yu cu phong ph, phc tp, a dng ca thc th
phn tch, t hiu sut cao v tit kim thi gian phn tch.

3. U D UV26

34

Hu ht phn tch sc k lng hin i s dng mt trong bn loi u d l u d UV,

u d dn in, u d hunh quang v u d pht hin ch s khc x. Trong u d
UV chim ti 80%.

3.1.

u d hp th UV

u d hp th UV nhy vi nhng cht hp th nh sng trong vng khong 180-350

nm. Hu ht cc cht hp th nh sng trong phm vi bc sng ny, bao gm c nhng cht c
mt hoc nhiu lin kt i (electron ) v cc cht khng c ni i (unbonded), v d tt c cc
olefin, tt c cc cht thm v cc hp cht cha nhm > C = O, > C = S, - N = N -. nh sng
UV sp xp i qua cc t bo v ri vo mt t bo quang in (hoc mng t bo quang
in). Tn hiu ra t cc t bo quang in c chuyn sang mt b khuch i sa i v sau
n mt h thng ghi hoc thu thp d liu.
Mi quan h gia cng ca nh sng UV truyn qua mt t bo (I T) v nng ca
cht tan cha trong t bo (C mol.L-1) c cho bi nh lut Lambert Beer.
IT = Ioe-klc
ln(IT) = ln(Io)-klc
A ln

IO
klc
IT

Trong (Io) l cng ca nh sng i vo t bo, (l) chiu di ca bung o,

(k) l h s hp thu phn t ca cht tan cho bc sng c th ca cc tia UV.
(A) hp thu quang
n v ca K ph thuc vo th nguyn ca l v C. Trong h SI th nguyn ca C l mol
m-3, (l) l mt nhng trong thc t C c o vi moldm-3 cn (l) l cm nn (k) l dm3mol-1cm-1.
Trch phn

35

d (log

IT
)
IO

dc

kl

nhy ca u d t l thun vi h s hp thu (k) v chiu di bung o (l). tng

nhy ca h thng, chiu di ca bung o (l) phi tng dn n th tch tng do chiu di
bung o (l) phi c hn ch. iu ny cn thit gim thiu phn tn ca pic trong cc
cm bin v trnh nhiu hn mt phn nh ca pic tn ti trong t bo ti bt c thi im
no. hn ch s phn tn pic chiu di bung o (l) c tng ln, bn knh ca bung o
gim. Nh vy, c t nh sng s ri vo photo-cell, t l signal-to-noise s b gim v do
nhy u d hoc nng pht hin ti thiu gim.
Do , vic tng nhy pht hin bng cch tng chiu di ng dn (l) c nhng hn
ch v bung o phi c thit k hi ha gia bn knh v chiu di cung cp nhy ti
a. Hu ht cc cm bin d UV hin i c chiu di ng dn t 1 n 10 mm v bn knh
trong khong t 0,5 n 2 mm.

Log

IT
Io

= klc = A

A = k' lc
(A) hp thu
(A) c dng xc nh nhy ca u d,
gi tr ca (A) l s thay i hp thu cung cp mt t l signal-to-noise.
(c) nhy nng u d hoc nng pht hin thp nht.
Do , hai u d c cng nhy ngha l c cng pht hin ti thiu, khng nht
thit phi c cng nhy nng cht tan. Ch cn di bung o ca hai cm bin ging ht
nhau th hai u d s cng nhy.
u d UV c th c s dng ch ra gii Gradient bng cc dung mi khng c
tnh hiu hp thu ng k trong di bc sng ang c s dng cho pht hin. Trong sc k
pha o, cc dung mi ra gii thng l nc, methanol, acetonitril v tetrahydrofuran (THF),
cc dung mi ny trong sut i vi nh sng UV trn tng phm vi bc sng thng c s
dng bi cc u d UV. Trong sc k pha thng cn thn trng hn trong vic la chn dung
36

mi. Nhiu dung mi c th ph hp vi h thng sc k c kh nng hp th nh sng UV rt

mnh m. Cc n-parafin, methylene dichloride, ancol bo v THF l dung mi thng dng v
trong sut trong tia UV v c th c s dng vi cc h thng phn b thng (v d mt pha
tnh phn cc nh
gel silica).
Trong detector UV-VIS chm sng a sc c cho i qua mu sau hi t vo khe vo
ca b n sc ha (monochromator) tch chm tia n sc cn o hng vo b d tm.
-

Mt quang ca mt mu c tm thy nh so snh gia cng ca chm tia ti

b

d tm khi khng c mu (cn gi l mu trng) vi chm tia i qua mu.

-

o mt quang ti nhng bc sng khc nhau, bc sng c thay i nh quay

chm lng knh hoc b cch t (grating) trong b n sc ha.

S hin din ca b phn quay trong thit b gii thiu thm sai s (v d do s git li

c hc) v vic quay chm c th gy tn thi gian.

-

Trong detector chui photodiot, chm tia a sc sau khi i qua mu c tn x bi b

cch t c c nh ri ri vo mt chui cc photodiot.

-

Mi photodiot o mt di hp cc bc sng trong quang ph, nh vy n l b thu nhn

s liu song song, ngha l tt c cc im c o ng thi. H thng ny c mt s thun li

nh sau:
-

Bi v l b thu nhn s liu song song nn vic x l v lu tr quang ph c th c

thc hin trong khong 0.5 s so vi vi pht trong detector UV.

V khng c b phn di chuyn c th b lm mi mn nn nhng sai s khi thit lp li

mt bc sng cn chn c gim xung v thit b cng t i hi bo dng nh thit b

khc.
-

Nh kh nng thc hin nhng php o nhiu bc sng cng mt lc v tc

thu nhn s liu nn nhng k thut ly trung bnh s liu khc nhau c th c s dng lm
gim ting n v tng nhy.
-

Khi c s dng nh mt detector trong HPLC, mc d t tin hn so vi loi UV

truyn thng nhng n cng c nhng u im ng k:

-

Quang ph ca mi pic trong sc k c th c lu gi v ln lt so snh

vi ph chun gip tin li trong vic nhn din cc pic.

37

Bc sng ti u cho vic d tm cng d dng c tm thy v nhng thay i

ca bc sng trong qu trnh d tm cng c th c chng trnh ha xy ra ti nhng

im khc nhau trong sc k .
-

N cng cung cp nhy cc i cho cc pic.

u d UV a bc sng 27
u d a bc sng s dng mt ngun sng pht ra nh sng qua mt lot cc bc
sng. S dng mt h thng quang hc thch hp (mt lng knh hoc cch t nhiu x), nh
sng c bc sng c th c la chn cho cc mc ch pht hin, ti cht tan cn phn tch
c hp thu cc i cung cp nhy ti a. Ngoi ra, quang ph hp thu ca cht ra gii
c th nhn dng bng cch qut qua mt lot cc bc sng.
C hai loi u d a bc sng c bn, u d phn tn v u d mng diode, u d
mng diode l ph bin hn. Tt c cc u d a bc sng yu cu ngun sng pht ra nh
sng rng nh n xenon hoc n deuterium, n deuterium l nht ph bin.
Hai loi u d a bc sng c nhng khc bit quan trng. Trong cng c phn tn,
nh sng b phn tn trc khi n i vo t bo cm bin nh sng v do hu nh nh sng l
n sc khi i qua bung o. Tuy nhin, nu nh sng ti l mt bc sng c th kch thch cc
cht tan pht hunh quang ti bc sng khc, khi nh sng ri trn t bo nh s cha nh
sng ti cng vi bt k nh sng hunh quang c th c to ra. Theo , nh sng c
gim st bi cc t bo nh c th khng n sc v nu c nh sng ca bc sng khc s lm
gim tnh cht tuyn tnh ca phn ng. Hiu ng ny s khng ng k trong hu ht cc
trng hp, nhng vi mt s vt liu hunh quang hiu ng c th l ng k. u d mng
diode hot ng kh khc bit. nh sng ca tt c cc bc sng c to ra bi n deuterium
i qua bung o v sau c phn tn qua mt mng diode. Nh vy, s hp thu ca cc
nhm bc sng ring bit c theo di lin tc mi diode. Tuy nhin, nh sng chiu vo
mt diode ring bit c th cha nh sng c to ra bi s kch thch hunh quang bng nh
sng c bc sng ngn hn. T bo c tip xc vi nh sng ca tt c cc bc sng pht ra
bi ngun sng v v vy s pht hunh quang c nhiu kh nng. Nh vy, o nh sng truyn

38

qua c th lin quan n nh sng hunh quang v quang ph hp thu thu c ca mt cht c
th l mt hnh thc suy thoi ca ng cong hp thu ng.
u d a bc sng l tng s l mt s kt hp ca c hai h thng u d phn tn
v u d mng diode. S sp xp ny s cho php mt chm nh sng n sc tht s i qua
u d v sau chm tia truyn s c phn tn mt ln na ln mt mng diode. Ch diode
cm bc sng ca nh sng ti s c s dng gim st vic truyn qua. Bng cch ny,
loi b c nh sng hunh quang, s hp thu ng s c o v cm bin n sc chnh xc
c th t c.
u im: La chn c nhiu bc sng o; tng cng nhy pht hin bng
cch chn bc sng m cht quan tm c hp thu mnh v cht nn trong hn hp khng
cho tn hiu hp thu.

3.2.

u d DAD26

u d mng diode (DAD) ra i sau nm 1990, da trn s pht quang ca mt mng

diode c bit khi n b chm sng tc ng vo. V nguyn tc u d DAD cng hot ng
tng t nh u d ph hp thu quang phn t vng ph UV-Vis. Mi u d thng c hng
trm diode pht quang, to thnh mt mng diode, c vng ph lm vic t 190-1100 nm v c
tnh khng gian ba chiu. V vy loi u d ny thu nhn c hnh khng gian ca pic hp thu
ca cht phn tch v nhiu pic ng thi.
u d mng diode c nhy v chn lc cao, tc o ln hn v c th o c
t 2 n 42 knh ng thi, chn sng o hp thu cho tng cht l d dng, hn ch c s
trng sng hp thu. Hin nay u d DAD c dng kh ph bin trong cc h my HPLC.
s dng u d DAD cho k thut HPLC th cn phi c my tnh v b phn mm chuyn dng.
u d DAD (Detector Diod Array) c kh nng qut chng ph nh tnh cc cht
theo hp th cc i ca cc cht. Photodiode l thit b bn dn c th chuyn i nh sng
thnh dng in. Dng in c to ra khi cc photon c hp th trong photodiode.
Nu php o c thc hin ti mt bc sng c nh, cc thnh phn ch c xc nh
t thi gian lu; do , mt s sai lch nh trong thi gian lu c th lm cho vic xc nh cc

39

thnh phn kh khn. Trong mt trng hp nh vy, cc DAD c th c s dng xc nh

cc thnh phn bng cch so snh quang ph.
3.2.1.

Nguyn l hot ng

u d mng diode cng s dng mt n deuterium hoc n xenon pht ra nh sng

trong di quang ph UV. nh sng t n c tp trung bng mt ng knh tiu sc sau
truyn qua bung o v ln mt cch t ba chiu. nh sng phn tn t cch t c sp xp ri
vo mt mng diode tuyn tnh.
nh sng t ngun pht x rng c chun trc ca mt h thng ng knh tiu sc sao
cho tng s nh sng i qua bung o vo mt cch t ba chiu. Bng cch ny, nh sng ca tt
c bc sng c to ra bi n s i qua mu. nh sng phn tn t cch t ri vo mt mng
diode. Mng diode c th cha hng trm diode v tn hiu t mi diode ra lin tc trn my tnh
v lu tr trn a cng. Vo cui qu trnh, cc tn hiu ra t bt k diode c th c la chn
v mt sc k c to ra s dng cc bc sng UV m ri vo diode c bit.
Tn hiu ra ca mt diode l ghi nhn trong thi gian thc v cho ra mt sc k thi
gian thc s. Bng cch ghi nhn thi gian ca mt pic ring bit, mt quang ph c th thu
c bng cch nh li t b nh u ra ca tt c cc diode da vo thi gian ring bit.
phn gii ca u d () s ph thuc vo s lng cc diode (n) trong mng v
phm vi bc sng c iu chnh (2 - 1). Do , phn gii ca u d mng diode s ph
thuc vo nh sn xut bn dn.
=

2 1
n

40

Hnh : u d mng diode

C hai u im chnh ca pht hin dy diode.

Th nht, n cho php la chn cc bc sng tt nht (s) phn tch thc t. iu ny
c bit quan trng khi khng c thng tin v hp thu phn t cc bc sng khc nhau.
u im ln th hai c lin quan n cc vn v tinh khit ca pic. Thng
thng, hnh dng pic trong chnh n khng tit l rng n thc s tng ng vi hai thnh phn
(hoc nhiu hn). Trong mt trng hp nh vy, hp th phn phi (rationing absorbance) ti
mt s bc sng c bit hu ch trong vic quyt nh cc pic i din cho mt hp cht n l
hay pic ca mt hn hp. Trong phn phi hp thu, hp thu c o ti hai hoc nhiu bc
sng v t l c tnh ton cho hai bc sng c chn. o ng thi ti mt s bc sng
cho php tnh ton cc t l hp th. nh gi c th c thc hin theo hai cch:
Trong trng hp u tin, t l bc sng la chn lin tc theo di trong khi phn
tch: nu cc hp cht di pic l tinh khit, tn hiu thu c l mt hm sng vung hnh ch
nht. Nu khng phm hm sng vung pic l khng tinh khit.
S khc bit u d DAD v u d UV-Vis
Hnh 2 cho thy mt h thng quang hc DAD. u d DAD khc vi u d UV-VIS
trong nh sng t n chiu trc tip ln cc t bo dng chy, nh sng i qua cc t bo dng

41

chy c phn tn bi cch t nhiu x v s lng ca nh sng phn tn c c tnh cho

mi bc sng trong cc dy photodiode.
So vi mt my d UV-VIS, DAD c nhng nhc im sau y: bt n ln v
lng nh sng nh; DAD cng nhy cm vi nhiu thay i khc nhau, chng hn nh s bt
n n, bi v nh sng chiu khng th nhn c. Tuy nhin, DAD gn y c ci thin
gim s khc bit ca n trong hot ng t u d UV-VIS.

Cc kt qu ca mt php o vi mt DAD cung cp cc chc nng tin li, bao gm

kim tra tinh khit ca pic v tm kim th vin, cng nh phn tch nh lng vi mt sc
k xc nh.
Mt tnh nng c bit ca mt s u d UV c bc sng bin i l thc hin ph
qut v ghi ph hp thu chnh xc ti mt lot cc bc sng vi pic sc k i qua bung
o. Mng diode cho bit thm mt chiu hng mi ca kh nng phn tch sc k lng v n
cho php nh tnh da trn cc thng tin thu c ngoi cch nh tnh bi thi gian lu gi.

3.2.2.

Kim tra tinh khit cht phn tch

u d diode c th c s dng kim tra tinh khuyt ca mt cht tan nht nh c th

hin trong hnh 31. Gim st sc k ti 274 nm c hin th phn di ca hnh 31. Da vo tn hiu ra
t u d ti nhng bc sng khc nhau v t l ng thi vi vic gim st sc k ti 274 nm. Nu cc
pic l tinh khit, t l hp thu hai bc sng (bc sng c la chn l 225 v 245nm) s khng thay
i trong sut qu trnh ra gii ca pic.

42

S trn trong hnh 31 cho thy t l ny c v trn cng mt quy m v n c xem l mt

pic hnh ch nht sch c quan st r rng khng nh tinh khit ca pic chlorthalidone. Cc bc
sng chn cung cp t l xc nhn s ph thuc vo c im hp thu UV ca cc cht quan tm v
cc tp cht c kh nng c mt nht.

Chlorthalidone c phn lp t mt vin thuc v c tch bi o (C18) trn mt ct 4,6 mm

I.D., di 3,3 cm, hn hp dung mi ra gii gm 35% methanol,
65% dung dch axit axetic (c cha 1% nc). Tc dng chy l 2 ml / pht.
Hnh 31. S thu c t u d mng Diode kim tra tinh khuyt ca pic

S dng u d mng diode kim tra tnh ton vn ca pic ra gii bng cch tch hng
lot cc hydrocarbon thm trong hnh 32. Cc pic ca tng cht xut hin r rng n l hp l
gi nh rng tt c cc nh l tinh khit

43

S tch c thc hin trn mt ct di 3 cm, ng knh 4,6 mm v

nhi vi pha tnh o C18, ng knh ht 3 m
Hnh : S phn tch ca mt s hydrocarbon thm
Tuy nhin, bng cch v cc t l hp thu

250 nm
255 nm

cho pic anthracene n tr nn r

rng rng ui pic cha tp cht bi v hnh dng hnh ch nht sch ca pic l khng c hin
th. Cc pic t l m t trong hnh 33 cho thy r rng s hin din ca mt tp cht trn pic
anthracene.

Hnh : ng cong lin h t l hp thu

250 nm
255 nm

v thi gian

Pic anthracene th hin trong hnh 34. xc nh tp cht nh t-butyl benzen mc

khong 5%.

44

Mt sc k c hin th c nm pic v quang ph thu c pic (a) v pic (b) na

bn tng ca pic, u mi pic v na bn xung ca mi pic (hnh 35). Pic khng tinh khit v
pic tinh khit c xc nh r rng.
45

Hiu sut ca c hai loi u d a bc sng l tng t nhau v nhy c trng

khong 1x10-7g/ml (t hn ng k so vi u d bc sng c nh) vi mt linear dynamic
range
khong 5000 v mt ch s phn ng t 0,97 n 1,03.

4. NHNG NGHIN CU V C NGI BN BNG HPLC

4.1.

Nui cy m t bo c Ngi Bn in vitro

S tn ti cc hp cht flavonoid c hot tnh sinh hc trong c v t bo nui cy

ca c Ngi Bn27
X l mu
Cc b phn ca cy Ngi Bn c s dng: c, m so v nui cy treo t bo. C
c ra di vi nc chy 30 pht, thi lt v sy kh. Thc hin nui cy m theo qui trnh
Yusuf et al. (2011). Mi th nghim c thc hin lp li 30 ln. M so ng knh 1.5 cm
c nui cy trong mi trng bt u v subcultural mi bn tun trong mi trng nhn
ging (Tan et al, 2005). Tc tng trng m so c tnh ton bng cch s dng
phng trnh m t bi Yokota (1999).
Chit v phn tch HPLC
C, m so v cc t bo nui cy c sy kh 38 C v nghin thnh bt. Mu bt (1,0
g) c ngm trong 100 ml Methanol trong 72 h v c lc qua giy lc Whatman s 1. Dung
dch sau lc em c quay (Buchi Rotavapor R-114). Cao (slurry) thu c chit lng lng mt
lng bng nhau ca Ethyl Acetate v Nc. Phn Ethyl Acetate em c quay, ghi khi lng v
ti ha tan trong Methanol vi mt t l l 1,0 mg chit xut n 0,2 ml methanol. Dung dch
ethanol ca cao chit c lc qua b lc 0,45 m PTFE (Sartorius 13 CR) trc khi tim
HPLC.
Cht chun flavonoid: alpinetin, pinocembrin, cardamonin, pinostrobin v panduratin A
Phn tch nh lng nm flavonoid trn h thng HPLC (Waters, M), W600E multisolvent
delivery system, W2489 UV/visible detector, W2707 auto sampler and in-line de-gasser.
-

Ct pha o: Chromolith RP-18e, (100 x 4.6 mm i.d.; 2 m) (Merck) provided with guard
cartridges RP-18e (5 x 4.6 mm i.d.; Merck).

46

Pha ng s dng: H dung mi gradient: 0.1% axit Phosphoric (A) and Acetonitrile (B)
(Merck). (0.5 pht, 80% A, 20% B; 4.5 pht, 65% A, 35% B; 5.0 pht, 40% A, 60% B;

8.0 pht, 100% B), tng thi gian chy 18 pht

Tc dng: 1.5 ml/pht.
Bc sng theo di: 285 v 330 nm

Th tch mu mi ln tim 0,02 ml v xc nh cc flavonoid c thc hin bng cch so

snh thi gian lu ca quang ph hp th vi nhng cht chun tham chiu. nh lng
alpinetin, pinocembrin, cardamonin, pinostrobin v panduratin A trong cao chit c thc hin
bng mt phng php hiu chun p dng trong phm vi ca 0,002-1,0 g. Cc s liu c thu
thp v x l bng phn mm Empower 2R v th hin nh g mi hp cht theo trng lng
kh (DW).

47

48

Phn b ca cc hp cht Flavonoid v dn xut Cyclohexenyl Chalcone

trong nhn ging truyn thng v nui cy In vitro [28]
X l mu
C Ngi Bn ti (Malaysia), ra sch vi nc v kh trong khng kh sau t
trn mt lp bng sch r chi mc cao t nht 1- 2 cm. phn tch thn phn ha hc trong
cc m khc nhau B. rotunda, gm: r con, thn r, shoot-base, maroon thn, cung l v l .Tt
c u c xt lt mng, kh trong khng kh v xay thnh loi bt mn, bo qun -80C.
phn tch metabolic profile trong cc m khc nhau B. rotunda, CPA ca 2-3cm chiu
cao v cy CP c lm sch v tch ra thnh su c quan: rootlets, rhizomes, shoot-base
rhizomes, maroon stems, leaf stalks, and leaves (Hnh 1) . Tt c u c xt lt mng, phi
kh v nghin thnh mt loi bt mn s dng mt loi bt blender. Bt c bo qun -80C
n khi s dng.
Chit v phn tch HPLC
Bt trn c ngm trong methanol qua m. Sau lc v c quay 35C thu c cao
methanol. Cao thu c phn tch (partitioned) bng ethyl acetate (EA) v nc vi khi lng
bng nhau. Mc ch loi b cc hp cht qu phn cc trong cao chic. Phn EA em ht
chn khng lm kh, ghi li khi lng ca cao th. Cao th c ha tan trong methanol mt
t l 1mg trong 30-60 ml methanol v sau lc thng qua mt b lc 0,45 m PTFE (c
Acrodisc 13 CR) trc khi phn tch bng sc k lng hiu nng cao (HPLC). Cht chun
flavonoid: alpinetin, pinocembrin, cardamonin, pinostrobin v panduratin A
H thng HPLC (Perkin Elmer)

49

Ct pha o: semipreparative column (Chromolith SemiPrep RP-18 endcapped 100

10mm column, pore size 2 m to 13nm (Merck, Germany), Perkin Elmer Series 200

Pump,
u d: DAD
Pha ng s dng: h dung mi gradient

80% nc - axit phosphoric v 20%

acetonitrile trong 0.5 pht, sau trn gradient tuyn tnh bt u vi 80% axit
phosphoric, gim dn xung 70% trong 5pht v gi trong 7 pht v 50% trong 20 pht
-

gi trong 4 pht, 20% trong 14pht. Mi ln chy l 50.5 pht.

Tc dng: 1.5 ml/pht.
Bc sng theo di: 285 v 330 nm

Mi mu chy 5 ln, nhng peak c lch chun t hn 5% s c ghi li. Cc hp cht

c nh lng bng cch so snh hp thu vi ngoi chun c xc nh trc.
Naringenin c thm vo mu nh l cht ni chun.

Biu hin ca gen lin quan flavonoid, 4 coumarate: coenzyme A ligase v ti

u ha cc iu kin nui cy sn xut chn lc cc flavonoid trong c Ngi
Bn [29]
X l mu
C Ngi bn mt nm tui c trng ti i hc Malaya, Malaysia . c thu thp lm sch
di vi nc trong 10 pht. C Ngi Bn sau khi lm sch c t vo ti nha chi ny
mm cao khong 24 cm trc khi s dng nh explants for initiating cell suspension culture.
For reverse transcription-real time PCR (RT-qPCR) analysis.
L khng bnh, r v thn r ti sau khi thu hoch c nghin thnh mn bt bng chy v
ci c mt nit lng
Chit v phn tch HPLC
L, thn r v r c ct nh v c sy kh (oven-dried). T bo nui cy c lc v
lm kh ngoi khng kh (air dried), nghim thnh bt mn. Khong 1g bt mn t mi mu
ngm trong methanol 100 % trong 72 h. Sau khi lc ting hnh c quay ui dung mi. Mt
lng bng nhau ca Ethyl Acetate (Fisher Scientific, USA) v Sodium chloride (100 ml) thm
vo v trn qua m in a separating funnel to obtain different fractions. 2g Sodium Sulphate
(Systerm-ChemAR, USA) thm vo the remaining phases followed by filtration through a filter
paper. C quay loi b dung mi. o ghi khi lng mi cao chit, thm mt lng Methanol

50

c c nng cao 1 mg/ml. Hn hp lc qua b lc 0.45 m PTFE (SartoriusR 13 CR, USA)

thu vo mt vial tube. Mu methanolic sau lc (20 l) np vo HPLC:
-

Ct: Chromolith performance RP-18e endcapped 1004.6 mm (Merck Milipore, USA),

WatersR 2707 Autosampler, WatersR 2489 UV/Visible Detector, and WatersR 600.
Tc dng: 1.5 ml/pht.
H dung mi: 80 % water-phosphoric acid v 20 % acetonitrile for 0.5 pht, sau trn

vi 80 % dn dn gim xung 65 % (hn 4 pht), 40 % over 0.5 pht, 0 % over 3 pht.

Bc song theo di: 285 v 330 nm.

3 flavonoid: panduratin A, pinocembrin v pinostrobin (thu t Department of Chemistry,

University of Malaya, Malaysia) chn lm cht chun vi nng 1 mg/ml ( Hnh 1).
Cc hp cht cn nghin cu c xc nh bng cch so snh t R ti 4.3, 9.1 v 10.6 pht
tng ng vi panduratin A, pinocembrin v pinostrobin. Mi mu chy 3 ln.
Tng khi lng flavonoid (g/g)
=

Th tch dch chit ( ml)

Din tch peak mu
x nng cht chun x
Din tch peak ca cc cht chun
Khi l
ngcao th( g)

51

Panduratin A c ch s tng trng ca t bo A549 qua cm

ng apoptosis v c ch NF-KappaB
chuyn v 30
Chit v phn tch HPLC
C Ngi Bn kh (4 g) (Malaysia) c chit xut hai ln vi methanol (50 ml) trong 48 gi
nhit phng, thay th khong cng mt lng Methanol mi sau 24 gi u tin. Cc chit
xut c lc qua polyvinylpyrrolidone loi b tannin trc kt hp v lm kh trong chn
khng c c chit th (0,4 g).
Cao th u tin c phn on s dng phn tch HPLC / LCMS-IT-TOF trn thit b
Shimadzu
-

Ct pha o: Waters Xbridge RPC18 column (2.1 x 50 mm, particle size 2.5 m)
u : PDA
Pha ng s dng: hn gradient gia acetonitrile v nc t 10 n 100% (v / v) trong 7

pht.
Tc dng: 0,5 ml / pht
Nhit ct: 40 C.

Th nghim sinh hc kh nng gy c ca cc phn on nm trong trong khu vc 6-7 pht

cha mt peak ln duy nht c xc nh l panduratin A da trn MS / MS. Panduratin A c
phn lp t dch chit th bng HPLC iu ch pha o.
-

Ct: (Waters Nova-Pak C18, kch thc ht 6 mm, 25 x 100 mm)

52

Pha ng: hn hp gradient Acetonitrile v Nc t 60% n 100% (v / v) Acetonitril

trong Nc
Tc dng: 12 mL / pht c p dng trn 50 pht.

nh tnh v tinh khit (> 98%) ca panduratin A c lp c xc nh bng phn tch

HPLC v (1H-NMR). 1H-NMR trong CDCl3 ging vi bo co trc y [24].

c tnh chng ung th ca panduratin A phn lp t c Ngi Bn4

Chit v phn tch HPLC
C Ngi Bn (Indonesia) kh thi lt (100 g) c ngm, trch vi ethanol tuyt i (250 ml)
qua m nhit phng. Chit xut ethanol sau c lc ht chn khng v chit ba ln. C
quay 35 C trong chn khng, thu c 13,6% cao kh. Cao ethanol th phn on bng sc
k lng iu ch (Prep Nova-Pak HR Prep LC column, particle size 6 lm, 200x25 mm; c
trang b vi mt ct bo v c cha mu) s dng hai dung mi ra gii khc nhau.
Phn A ra gii vi 1: 1 methanol/nc c cha 0,025% (v / v) axit trifluoroacetic (TFA).
Phn B ra gii vi 100% methanol. Sc k lng c chy vi tc dng 20 ml / pht.
Panduratin A c phn lp t phn B bng sc k lng pha o s dng mt pha ng
gm mt gradient tng bc 60, 70, 75 v 80% (v / v) dch methanol cha 0,025% (v / v) TFA.
Panduratin A c lp c sau c kim tra tinh khit bng phn tch HPLC. H thng
HPLC gm mt Microsphere (Supelco, USA):
-

Ct: RP C18 (Kch thc ht 5 lm, 250 x 3,2 mm) c trang b vi mt ct bo v c

cha cc vt liu tng t.

Pha ng: chy ch gradient nng 0,25% axit axetic v 100% acetonitrile
Tc dng: 0,77 ml / pht
Nhit ct: 48 C.

Hot tnh bo v ca Panduratin A chng s xy ho gy ra bi

thioacetamide: chng minh trong th nghim in vitro bng dng t
bo gan WRL-68 31
X l mu
C Ngi Bn ti (Malaysia) ra vi nc my sau ra li vi nc ct, thi lt v phi
kh 10 ngy, xay thnh bt. 100 g bt c trch trong 1000 mL ethanol 95% trong 7 ngy
53

nhit phng. Hn hp c chng ct v sy kh 40 C trong 3 ngy thu c cao chit c

Ngi Bn 9,49% (w / w).
C lp PA t cao chit c Ngi Bn
2,67 g cao chit ha tan trong Axeton v trn vi 5 g gel silica, c quay cho kh. Bt thu
c em sc k ct (CC) cha 47 g gel silica. Cc phn on/ c lp l da trn phng php
ra gii Gradient vi h dung mi Hexane-Ethyl Acetate.
Tng cng c 11 phn s cng. Sc k lp mng ca 11 phn on so vi cht chun PA
(98% tinh khit) l thc hin theo t l 95: 5. Phn BR-3 c cha PA (Rf 0,29) c sy
kh bng cch c quay thu c 0,767 g tinh th PA tinh khit (96,6%) c xc nhn bng
(HPLC) v c tnh bng cng thc sau:
DintchpeakpanduratinA
% tinh khuyt = t(
ngdi

chpnteackp
hdetniad
ku
nm
g)i

Hot ng ca panduratin A c lp t c Ngi Bn Against multispecies oral biofilms in vitro 32

Chit v phn tch HPLC
C Ngi bn kh thu thp Jakarta, Indonesia, c xc nh bi N. I. Baek, Department
of Oriental Medicinal Materials and Processing, Kyunghee University (Yongin, Korea).
Kaempferia pandurate Roxb. (100g) c chit vi 95% Ethanol (400ml) thu c 11.95g cao
kh. Chit vi Ethyl acetate (2 x 200ml). Chy sc k ct silicagel (5 x 45 cm, 650 g silica gel;
70-230 mesh; Merck KGaA, Darmstadt, Germany), ra gii vi n-hexane-chloroform-ethyl
acetate 15:5:1.5 (21, v/v/v) thu c 7 phn on(1-7).
Phn on 3 (0.91-11,1.51 g) tip tc tch vi n-hexane-ethyl acetate-methanol 18:2:1
(1.51, v/v/v), thu c phn on 3-B (1.1 g). Phn on 3-B ra gii vi Methanol 100% (0.80.9 ml) phn tch HPLC iu ch:
- Ct: JAIGEL W-252 column, 20.0 mm i.d. x 500 mm L; Japan Analytical Industry Co.,
-

Ltd., Tokyo, Japan)

u d: UV
Bc sng hp thu: 365nm
Pha ng s dng: 100% Methanol
Tc dng: 3 ml/ pht
54

Thu c PA (300-400ml, 0.9g, 98%)

Phn tch kt cu ca mt hp cht Novel antimutagenic,

4-Hydroxy Panduratin A, v cc hot tnh antimutagenic ca cc
Flavonoid trong c Ngi Bn chng li t bin d vng Amin7
Chit v phn on cc hp cht chng gy t bin
C Ngi Bn ti (1,0 kg) c ngm, trch trong methanol (3.0 L) nhit phng trong
vng 24 h. Dch chit c quay 35 C, cao methanol thu c chit lng-lng vi Diethyl ether
(250 ml) v nc (100 ml). Cao Diethyl ether, c hot tnh antimutagenic, tin hnh sc k trn
ct ca Wakogel LP-40 C18 (5,0 x 20 cm), ra gii gradient tng bc: 40, 50, 60, v 70%
ethanol / nc, 0,1% axit formic, 1.5 L tng bc.
Cc phn on c hot tnh sinh hc tin hnh HPLC iu ch (TSK gel ODS-80Ts, 20 x
250 mm) tinh khit hn. Cc phn on hot tnh 1-6 c kt tinh trong methanol (60-80%).
Cc hp cht 1-6 tinh khit v ng nht (> 99% bi HPLC) v xc nh l antimutagens.
Xc nh cc hp cht trong 1,0 kg c BR thu c 1-6 : 249 mg, 801 mg, 270 mg, 816
mg, 39 mg, v 157 mg. Cu trc ca cc hp cht 1-6 (Hnh 1).

nh lng s b cc hp cht 1-6 trong BR

Phn tch HPLC: s dng h thng phn tch HPLC
-

Ct: TSKgel super-ODS (4.6 x 100 mm).

Pha ng: Gradient tuyn tnh s dng 0,5% axit formic v acetonitrile

u d: DAD

Nhit ct: 40 C.

Tc dng: 1,0 ml / pht.

Khi lng tim: 10 l

Bc sng theo di: 280 v 340 nm.

X l mu HPLC
C BR ti (10g) t 16 mu ring bit c chit trong 90 ml methanol 30 pht. Mt phn
chit xut c lc (mng 0.22 m), dch lc c s dng phn tch. Cc hp cht tinh khit
1-6 c s dng nh l cht chun. nh lng c tnh ton t cc ng cong hiu
chun ca cc hp cht 1-6 da vo nng tnh bng h s dp tt phn t ca chng.
55

Khi lng trung bnh cc hp cht (mg/100 g c Ngi Bn ti)

Cht
Khi lng

1
29 18

2
78 52

3
56 16

56

4
118 53

5
7.8 4.1

6
30 16

5. THM NH PHNG PHP33

5.1.

nh ngha

Thm nh phng php l s khng nh bng vic kim tra v cung cp bng chng
khch quan chng minh rng phng php p ng c cc yu cu t ra. Kt qu ca
thm nh phng php c th c s dng nh gi cht lng, tin cy ca kt qu phn
tch. Thm nh phng php phn tch l mt phn khng th thiu nu mun c mt kt qu
phn tch ng tin cy.
nh tr phng php, nh gi phng php, xc nhn gi tr s dng ca phng php,
ph duyt phng php l nhng cch gi khc nhau ca thm nh phng php (method
validation).
Da vo ngun gc c th phn loi cc phng php thnh hai nhm:
- Cc phng php tiu chun: cc phng php th theo tiu chun quc gia, quc t, hip hi
khoa hc c chp nhn rng ri trn th gii nh TCVN, ISO, ASTM, AOAC
- Cc phng php khng tiu chun hay phng php ni b (nonstandard/ alternative/in-house
method): l cc phng php do phng th nghim t xy dng, phng php theo hng dn
ca nh sn xut thit b, phng php theo cc tp ch, ti liu chuyn ngnh...
Theo yu cu ca ISO 17025, phng php phn tch phi c thm nh hoc thm
nh li khi:
- Phng php p dng khng phi l phng php tiu chun (nonstandard method)
- Phng php do phng th nghim t xy dng mi trc khi a vo s dng thnh thng
quy.
- C s thay i v i tng p dng nm ngoi i tng p dng ca phng php thm
nh hoc phng php tiu chun.
- C s thay i cc iu kin thc hin phng php c thm nh (v d: thit b phn
tch vi cc c tnh khc bit, nn mu, ngi phn tch).

5.2.

Ch tiu thm nh

Theo cc quy nh ca USFDA, AOAC, USP v ICH, i vi cc phng php phn tch
ha hc cc thng s cn thm nh bao gm:
- Tnh ph hp h thng (System suitability determination)
57

H thng ph hp l nh gi ca cc thnh phn ca mt h thng phn tch cho thy

rng hiu sut ca mt h thng p ng cc yu cu tiu chun ca mt phng php. Mt nh
gi s ph hp h thng thng cha cc thng s thit lp ring. i vi xt nghim sc k,
nhng thng s ny c th bao gm cc tailing factors (yu t i xng), resolution, and
precision of standard peak areas, and comparison to a confirmation standard, capacity factors,
retention times, and theoretical plates. Trong qu trnh thm nh, khi p dng, cc thng s h
thng ph hp l c tnh ton, ghi chp, v xu hng trong sut qu trnh thm nh. Gi tr
cui cng sau c xc nh t lch s ny (During validation, where applicable, system
suitability parameters are calculated, recorded, and trended throughout the course of the
validation. Final values are then determined from this history).
- Khong xc nh (range) l khong gia cc nng trn v di (amounts) ca cht
phn tch trong mu (bao gm c nhng nng ny), n c chng minh rng quy trnh
phn tch c mt mc ph hp ca ng, chnh xc v tuyn tnh.
Khong xc nh thng c th hin trong cc n v tng t nh kt qu xt nghim
(v d, phn trm, phn triu) thu c bng phng php phn tch.
Sut qu trnh thm nh, khong xc nh (i khi c gi l khong tuyn tnh ca
phng php) l nh gi theo mu (usually spiked placebos) v phi bao gm cc c im k
thut khong xc nh ca cc thnh phn kho nghim trong cc sn phm thuc.
- Tnh c hiu, tnh chn lc; (Specifility/Selectivity)
- Khong tuyn tnh v ng chun; (Linearity and Calibration curve)
- Gii hn pht hin; (Limit of Detection LOD)
- Gii hn nh lng; (Limit of Quantitation LOQ)
- ng; (Trueness)
- chm; (Precision)
- vng (n nh) ca phng php; (Robustness/Ruggedness)
Vic la chn cc thng s thm nh ty thuc vo k thut p dng trong phng php,
yu cu ca phng php, iu kin v ngun lc ca phng th nghim... Tng trng hp c
th cc thng s thm nh c th c s khc nhau.
Mi phng php phn tch ha hc c mt mc ch nht nh, t vic xc nh mt
cht nng thp n xc nh tinh khit ca mt cht. d dng cho vic la chn cc
58

thng s thm nh, cc phng php c th c chia lm bn nhm ty thuc vo mc ch

ca chng:
- Cc phng php phn tch nh tnh;
- Cc phng php phn tch vi lng;
- Cc phng php phn tch a lng;
- Xc nh gii hn tp cht (th tinh khit) (quang trng).
Cc yu cu v thng s cn thm nh cho tng loi phng php c m t bng 1
(phng php khng tiu chun) v bng 2 (phng php tiu chun). Ngi thc hin thm
nh cn la chn ng cch thm nh cho tng thng s v lp k hoch cho tng ni dung.
Bng 1: La chn thng s thm nh phng php khng tiu chun
Phn tch nh

Phn tch vi

Phn tch a

Xc nh gii

tnh

lng

lng

hn tp cht

(Specification/Selectivity

)
LOD (Limit of detection)
LOQ

+/-

+/-

+
+
(Ruggedness/Robustness)
(-) khng cn thc hin thm nh; (+) cn thc hin thm nh

Cc thng s thm nh
ng
(Trueness)
chm
(Precision)
c hiu, chn lc

(Limit of quantitation)
tuyn tnh
(Linearity/range)
vng (n nh)

Bng 2: La chn thng s thm nh phng php tiu chun

Cc thng s thm nh
ng
(Trueness)
chm
(Precision)

Phn tch nh

Phn tch vi

Phn tch a

Xc nh gii

tnh

lng

lng

hn tp cht

59

 c hiu, chn lc
(Specification/Selectivity

)
LOD (Limit of detection)
LOQ

(Limit of quantitation)
tuyn tnh
(Linearity/range)
vng (n nh)

(Ruggedness/Robustness)
(-) khng cn thc hin thm nh; (+) cn thc hin thm nh

5.2.1.
5.2.1.1

Tnh c hiu/chn lc
nh ngha

Tnh c hiu: L kh nng pht hin c cht phn tch khi c mt cc tp cht khc

nh cc tin cht, cc cht chuyn ha, cc cht tng t, tp cht... Trong php phn tch nh
lng, l kh nng xc nh chnh xc cht phn tch trong mu khi b nh hng ca tt c cc
yu t khc, nhm hng n kt qu chnh xc. Tnh c hiu thng lin quan n vic xc
nh ch mt cht phn tch.
Tnh chn lc: L khi nim rng hn tnh c hiu, lin quan n vic phn tch mt s
hoc nhiu cht chung mt quy trnh. Nu cht cn xc nh phn bit r vi cc cht khc th
phng php phn tch c tnh chn lc. Nh vy, tnh chn lc c th bao trm c tnh c hiu.
Do cc phng php phn tch thng c nhiu cht cng xut hin nn khi nim tnh chn lc
thng mang tnh khi qut hn.

5.2.1.2

Cch xc nh

Trng hp chung
xc nh tnh c hiu/chn lc ca phng php nh tnh, nh lng cn b tr cc
th nghim nh sau:
- Phn tch cc mu trng, lp li ti thiu 6 ln i vi tng loi nn mu. Mu trng
phi khng c cho tn hiu phn tch. Nu mu trng c hn 10% dng tnh hoc xut hin
tn hiu th cn phi thay i phng php loi tr cc nh hng.

60

- Phn tch mu th hoc mu trng thm chun hm lng gn LOQ, lp li ti thiu 6

ln. So snh kt qu vi mu trng, phi cho tn hiu cht cn phn tch.
- S dng phng php thm chun sau chun b mu (cochromatography), cch ny
thng p dng i vi cc phng php sc k. Sau khi chun b mu (mu trng hoc mu
thc) v phn tch mu trn thit b sc k thu c cc pic sc k, ta thm chun vo mu
chit xut v phn tch mu ny. So snh sc k ca hai mu nh gi tnh c hiu/chn
lc.
- Phn tch mu khng c cht phn tch nhng c cht cu trc tng t cht phn tch (nu c):
Phi cho kt qu m tnh (i vi phng php nh tnh) v khng c nh hng n kt qu
nh lng ca cht phn tch (i vi phng php nh lng). Trong trng hp nhng ch
tiu phn tch khng th c mu trng (sample blank) xc nh tnh chn lc/c hiu, c th
thc hin cc th nghim trn cc mu trng thuc th (reagent blank), tc l thc hin phn tch
cc bc tng t nh khi phn tch mu nhng khng c mu th.
Cc trng hp c bit
Sc k lng s dng detector DAD (mng diod)
Tnh chn lc trong sc k lng c th t c thng qua vic la chn ct ti u, iu
kin sc k ti u, nhit ct v bc sng. Ngoi vic thay i iu kin sc k, th giai on
x l mu cng phi ti u t c tnh chn lc cao nht. Trong sc k, cn xc nh rng
pc sc k c phi l mt pc n hay c ln cc tp cht khc. i vi cc h thng sc k c
detector mng diod (DAD) c th xc nh c tnh chn lc thng qua xc nh tinh khit
(pic purity) ca pc, hay so snh ph ca pc vi th vin ph sn c.

61

Hnh 1: Xc nh tinh khit ca pic trong sc k lng HPLC-DAD

tinh khit ca pc c xc nh bng cch so snh ph ti cc im khc nhau trn
pic sc k. Cch ph bin nht l chn ti ba im: nh pc (apex), v hai im v hai bn sn
ca pc (upslope v downslope). Thng thng chn hai im ti 2/3 rng ca pc v hai pha.
Ngoi ra hin nay a s cc thit b u c phn mm tnh ton tinh khit ca pc, c
th da vo 3 im, 5 im, 7 im hay tt c cc im to thnh pc. Cc thit b hin i c th
tnh ton tinh khit trc tip thng qua phn mm iu khin.

62

Hnh 2: Cc cch xc nh tinh khit ca pic sc k HPLC-DAD

So snh ph ca pic vi ph chun cng l mt cch ph bin xc nh s tinh khit
ca pc. Mt pc c xem l khng tinh khit khi gi tr ph hp (h s match) khng t xp x
100%. Tuy nhin, nu gi tr ny c gn 100% cng khng th khng nh c chc chn s
tinh khit ca pc, c th do mt trong s cc nguyn nhn sau:
- Tp cht tn ti vi lng thp hn nhiu so vi cht phn tch do ph UV-Vis
khng nh hng nhiu n ph ca cht phn tch.
- Ph ca tp cht v cht phn tch tng t nhau.

5.2.1.3

Tnh c hiu/chn lc i vi phng php chun

i vi cc phng php chun, nh nu trong bng 2, thng thng khng cn xc

nh tnh c hiu/chn lc. Tuy nhin, cn cn nhc thc hin vic xc nh c hiu trong
cc trng hp sau:
- Nn mu phn tch ti phng th nghim khc vi nn mu vi mu nu trong phng
php tiu chun. Trong trng hp ny cn thc hin y nh khi thm nh phng php ni
b.
- C s khc nhau v thit b phn tch m s khc nhau ny c th nh hng n tnh
chn lc. C th thc hin y hoc xc nh nh hng nu c thng qua xc nh hiu nng
ca thit b.
63

5.2.2.

Khong tuyn tnh v ng chun

Khong tuyn tnh ca mt phng php phn tch l khong nng c s ph

thuc tuyn tnh gia i lng o c v nng cht phn tch. Khong lm vic ca mt
phng php phn tch l khong nng gia gii hn trn v gii hn di ca cht phn tch
(bao gm c cc gii hn ny), ti c chng minh l c th xc nh c bi phng php
nht nh vi ng, chnh xc v tuyn tnh nh nu.
n gin hn, hai khi nim ny c m t trong hnh 3 di y:

Hnh 3: Khong tuyn tnh (linear range) v khong lm vic (working range)

5.2.2.1

Cch xc nh khong tuyn tnh

i vi hu ht cc phng php nh lng, cn phi thc hin vic xc nh khong

tuyn tnh. Vic xc nh khong tuyn tnh thng c kho st bt u t gii hn nh lng
(im thp nht) v kt thc l gii hn tuyn tnh (im cao nht). Ni chung, xc nh
khong tuyn tnh cn khong 10 (ti thiu l 6) nng khc nhau.
xc nh khong tuyn tnh cn thc hin o cc dung dch chun c nng thay i
v kho st s ph thuc ca tn hiu vo nng . V ng cong ph thuc gia tn hiu o v
nng , sau quan st s ph thuc cho n khi khng cn tuyn tnh. Khong tuyn tnh di
hay ngn ph thuc vo nhiu yu t, trong quan trng nht l bn cht ca cht phn tch v
k thut s dng. Cc cht khc nhau c khong tuyn tnh khc nhau do s khc nhau v tnh
64

cht l ha. Trong khi cc k thut s dng khc nhau nh hng ln n di ngn ca
khong tuyn tnh. V d, k thut HPLC nu s dng detector UV-Vis hoc DAD c th cho
khong tuyn tnh n 106 thm ch n 107, trong khi nu s dng detector hunh quang hay
ch s khc x th ch cho khong tuyn tnh khong 104 105.

5.2.2.2

Xy dng ng chun

Sau khi xc nh khong tuyn tnh cn xy dng ng chun v xc nh h s hi quy

tng quan. Trong phn tch thc t, c th xy dng cc ng chun ngn, trm ln vng nng
trong mu, khng nht thit phi lp ng chun ton b khong tuyn tnh. Nng trong
mu khng c vt ra ngoi gii hn cao nht v thp nht ca ng chun v tt nht
phi nm vng gia ng chun. C nhiu loi ng chun khc nhau ty thuc vo cc
phng php v k thut khc nhau, sau y l cc loi ng chun ch yu:
ng chun vi chun tinh khit
Chun b dy nng chun (ti thiu 6 nng ). Xc nh cc gi tr o c y theo
nng x (lp li 2 ln ly gi tr trung bnh). Nu s ph thuc tuyn tnh, ta c khong kho
st ng biu din l mt phng trnh:
y = ax + b
Trong : a: gi tr dc slope; b: gi tr h s chn intercept
R

( x X )( y Y )
(x X ) ( y Y )
i

v h s tng quan:

Nu 0,995 < R 1: C tng quan tuyn tnh r rt.

C th kho st tuyn tnh da vo tnh h s p ng ( th di). Khong tuyn tnh nm
trong khong th nm ngang.
Hnh cch lp ng chun tuyn tnh.
ng chun trn mu trng
Phn tch cc mu trng thm chun vi nng khc nhau (t nht 6 nng ), trong
khong tuyn tnh c lng trn, mi nng lm 3 ln. V ng cong ph thuc gia tn
hiu o (trc tung y) ph thuc vo nng (trc honh x). Tnh cc h s hi quy (a, b trong
phng trnh hi quy y = ax + b) v h s tng quan (R) tng t nh trn.
ng chun xy dng trn nn mu trng thng cho tin cy cao hn khi xy dng
vi chun tinh khit, do c th loi tr phn no cc nh hng ca nn mu. Tuy nhin, trong
65

nhiu trng hp kh tm c mu trng ph hp v khng c c cht phn tch. Do , c

th s dng phng php lp ng chun trn nn mu thc nh sau.
ng chun trn mu thc
Phn tch mu thc c cho thm cc nng chun khc nhau tng t nh trong phn lm vi
mu trng. V ng cong tn hiu o (trc tung y) ph thuc vo nng chun thm (trc
honh x). Dng ng chun trn nn mu thc thng c dng nh hnh 5:

Hnh 5: ng chun trn nn mu thc

Khi s dng ng chun trn nn mu thc c th loi tr c cc nh hng ca nn
mu n kt qu phn tch. Sau khi lp c phng trnh ng chun y = ax + b, c th d
dng tnh c nng : X = b/a
ng chun c s dng ni chun
Mt phng php rt hu ch trong phn tch, c bit trong phn tch hin i l s dng
ni chun. Ni chun c thm vo dung dch chun o my, vi nng ph hp v ging
nhau (CIS). V ng cong ph thuc gia t l tn hiu cht ngoi chun chia cho ni chun
(trc tung y) ph thuc vo nng ngoi chun (trc honh x). Tnh cc h s hi quy (a, b
trong phng trnh hi quy y = ax + b) v h s tng quan (R) tng t nh trn.

66

Hnh 6: V d ng chun s dng cht ni chun

Khi phn tch mu, ni chun cng phi c thm vo (tt nht l t u, sau khi cn,
ong) sao cho to c nng cui cng bng nng ni chun trong cc dung dch
chun. Vi cch tin hnh nh th ny c th hn ch c hu ht cc nh hng trong qu
trnh phn tch (bao gm t cn mu, chun b mu n phn tch trn thit b) n kt qu phn
tch.
i vi cc k thut phn tch hin i nh khi ph, c bit l sc k lng khi ph,
vic s dng ni chun l mt yu cu tin quyt, nu khng mun ni l bt buc. Thng
thng trong sc k lng khi ph, cc cht ni chun c u tin la chn l cc cht ng v,
thng thng nh 2H (D), 13C, 15N... V d, khi phn tch chloramphenicol th ngoi cht ngoi
chun l chloramphenicol, s dng thm cht ni chun l chloramphenicold5; clenbuterol th s
dng clenbuterol-d9, melamin th s dng melamin-13C15N... Cc cht ng v ny c u im
ni bt l tnh cht ha l gn nh tng t cht phn tch do i din tt cho cht phn tch
trong c
qu trnh. Ngoi ra, trong mt s trng hp c th chn cc cht ni chun khc, vi iu kin
l cc cht ny phi c mt s tnh cht c bn ging cht phn tch, v c th phn tch c
bng phng php ang thc hin.
Cc lu khi xy dng ng chun
- Cn m bo nng chun chnh xc: ng chun l yu t sng cn, quyt nh s
ng n ca kt qu phn tch, do nu trong qu trnh xy dng ng chun mc nhng sai
67

s ln s dn n s mt chnh xc ca kt qu. iu u tin kim sot c s chnh xc

ca cc nng chun khi xy dng ng chun l cn m bo chnh xc ca cht chun
(cht chun mua t nh sn xut) v hm lng, tinh khit. Theo yu cu ca ISO 17025, cc
cht chun khi s dng cn c chng nhn ca nh sn xut v vn cn hn s dng. Trong mt
s trng hp, c th b tr cc th nghim nh gi cht lng cc cht chun trc khi a
vo s dng.
- Tn hiu cc ln o ca mi nng phi c lp li t yu cu: Khi thm nh
phng php, mi nng cn c o vi ln (ba ln) kim tra lp ca cc nng
chun.
- Loi tr sai s th nu cn thit: Mt s trng hp, c th gp cc sai s th (ngu
nhin) xut hin dn n vic ng chun khng p ng yu cu. Trong trng hp ny, c th
cn nhc loi tr cc im ny m bo s chnh xc.
Gii hn chp nhn ca ng chun
- H s hi quy tuyn tnh (R): Ch tiu u tin ca mt ng chun t yu cu l h
s tng quan hi quy (Coefficient of correlation). R phi t theo yu cu sau:
0,995 R 1
Hay

0,99 R2 1

- chch cc im nng dng xy dng ng chun. Sau khi lp ng chun

xong cn kim tra bng phng php tnh ngc li nng ca cc im chun s dng xy
dng ng chun, t tnh cc gi tr chch theo cng thc sau:
i

Trong :

Ct CC
100
CC

: chch ca tng im chun dng xy dng ng chun

Ct : Nng tnh ngc theo ng chun ca cc im chun

Cc : Nng ca cc im chun
Theo quy nh ca nhiu t chc ca M, Canada, chu u, gi tr

khng c vt qu 15% cho tt c cc nng , ring nng LOQ c th chp nhn gii
hn 20%.

68

5.2.3.

Gii hn pht hin (LOD)

Gii hn pht hin l nng m ti gi tr xc nh c ln hn khng m bo

o ca phng php. y l nng thp nht ca cht phn tch trong mu c th pht hin
c nhng cha th nh lng c (i vi phng php nh lng).

5.2.3.1.

Cch xc nh

LOD ca phng php nh tnh

Cn xc nh c nng no m ti s xc nh chc chn s c mt ca cht phn
tch. Phn tch cc mu trng thm chun cc nng nh khc nhau, mi nng phn tch
lp li 10 ln. Xc nh t l phn trm s ln pht hin (dng tnh) hoc khng pht hin (m
tnh).

LOD ca phng php nh lng

C nhiu cch xc nh LOD khc nhau ty thuc vo phng php p dng l phng php
cng c hay khng cng c. Cc cch tip cn c th chp nhn c bao gm:
Da trn lch chun
Cch 1. Lm trn mu trng (mu trng c thnh phn nh mu th nhng khng c cht phn
tch).
Phn tch mu 10 ln song song, tnh lch chun. lch chun ny phi khc 0.
Tnh LOD:

LOD =
SD0

x0

+ 3SD0

(x

Vi
Trong :

x0

0i

x0 ) 2

n 1

nng trung bnh mu trng

SD0 lch chun ca mu trng

Cch 2. Lm trn mu th: Lm 10 ln song song. Nn chn mu th c nng thp (v d,
trong khong 5 n 7 ln LOD c lng).
Tnh LOD: Tnh gi tr trung bnh

, v lch chun SD
LOD = 3 x SD

69

SD0

nh gi LOD tnh c: tnh R =

(x

0i

x )2

n 1

/ LOD

Nu 4 < R < 10 th nng dung dch th l ph hp v LOD tnh c l ng tin cy

Nu R < 4 th phi dng dung dch th m c hn, hoc thm mt t cht chun vo

dung dch th dng v lm li th nghim v tnh li R

Nu R > 10 th phi dng dung dch th long hn, hoc pha long dung th dng v
lm li th nghim v tnh li R

Da trn t l tn hiu trn nhiu (S/N)

Cch ny ch p dng i vi cc quy trnh phn tch s dng cc cng c c nhiu
ng nn. Thng thng cch tnh ny p dng ph bin cho cc phng php sc k, in di.
Phn tch mu (mu thc, mu thm chun hoc mu chun) nng thp cn c th
xut hin tn hiu ca cht phn tch. S ln phn tch lp li 4 ln. Xc nh t l tn hiu chia
cho nhiu (S/N = Signal to noise ratio),
trong

S l chiu cao tn hiu ca cht phn tch,

N l nhiu ng nn

Nhiu ng nn c tnh v hai pha ca ng nn v tt nht l tnh nhiu ln cn

hai bn ca pc, b rng mi bn ti thiu gp 10 ln chiu rng ca pc ti na chiu cao.
LOD c chp nhn ti nng m ti tn hiu ln gp 2-3 ln nhiu ng nn, thng
thng thng ly S/N =3

Hnh 7: Xc nh LOD bng cch tnh S/N

70

Da trn ng chun (tham kho)

Ch p dng c cho cc phng php c xy dng ng chun.
LOD c th c xc nh da vo dc ca ng chun v lch chun ca tn
hiu o.
LOD

Trong :

3.3 SD
a

SD: lch chun ca tn hiu

a: dc ca ng chun

Gi tr a c th d dng tnh c t ng chun, gi tr SD c th c tnh theo nhiu cch

khc nhau, bao gm:
- Da trn lch chun ca mu trng: Phn tch mu trng lp li 10 ln v tnh SD tng
ng;
- Da trn lch chun ca mu thm chun nng nh gn LOD, lp li 10 ln (xem phn
kim tra pha trn) v tnh SD;
- Da trn h s chn ca ng chun, lm nhiu ln tnh SD ca
gi tr b (intercept);
- Da trn lch chun ca khong cch cc gi tr o thc vi ng chun.

5.2.4.

Gii hn nh lng (LOQ)

Gii hn nh lng l nng ti thiu ca mt cht c trong mu th m ta c th nh

lng bng phng php kho st v cho kt qu c chm mong mun. LOQ ch p dng cho
cc phng php nh lng. Ging nh LOD, c nhiu cch khc nhau xc nh LOQ, ph
thuc vo tng phng php c th m la chn cho ph hp. Vic xc nh LOQ cn tnh n
cc yu t nh hng trong mu phn tch, do cn thc hin trn nn mu tht.
LOQ trong nhiu trng hp c th l im thp nht ca khong tuyn tnh. Hnh 8 m t mi
quan h gia LOD, LOQ v khong tuyn tnh.

71

Hnh. mi quan h gia LOD, LOQ v khong tuyn tnh

4.2. Cch xc nh
Vic b tr th nghim xc nh LOQ thng kt hp vi tnh LOD.
C nhiu cch khc nhau tnh LOQ nh sau:
Da trn lch chun:
C hai trng hp nh trong phn tnh LOD l thc hin trn mu
trng v thc hin trn mu th. Cc cng thc tnh ton nh sau:
Tnh trn mu trng:
Tnh trn mu th:

LOD x0 10SD0

LOD 10SD0

Da trn t l tn hiu trn nhiu:

Cch ny ch p dng i vi cc quy trnh phn tch s dng cc cng c c nhiu
ng nn. Cch tnh ton hon ton tng t nh trong phn tnh LOD.
LOQ c chp nhn ti nng m ti tn hiu ln gp 10-20 ln
nhiu ng nn, thng thng thng ly S/N = 10.
Da trn ng chun:
Cch tnh tng t nh trong phn LOD nhng theo cng thc sau:
LOD

10SD
a

72

Lu , ngoi vic tnh ton nh trn cn quan tm n lp li ti LOQ, ty theo mi

phng php, gi tr RSD% cn phi t c theo mt mc yu cu no .

5.2.5.

chnh xc ( ng v chm)

Hin nay c nhiu cch hiu khc nhau v thut ng chnh xc. Trc y v n by
gi nhiu ti liu c ni v ng v chnh xc nh l hai khi nim khc nhau. Trong ti
liu ny chng ti s dng hai thut ng ng v chm din t chnh xc ca mt
phng php phn tch theo quan im mi nht ca tiu chun quc t (ISO 5725 1- 6:1994) v
tiu chun quc gia (TCVN 6910 1-6:2005). ng ch mc gn nhau gia gi tr trung
bnh ca kt qu th nghim v gi tr thc hoc gi tr c chp nhn l ng . chm ch
mc mc dao ng ca cc kt qu th nghim c lp quanh tr gi trung bnh. Hnh 9
m t s mi quan h gia chm, ng v chnh xc.
chnh xc (accuracy) = chm (precision) + ng (trueness)

5.2.5.1. chm
Trong nhiu trng hp cc php th nghim trn nhng i tng v vi nhng iu
kin khc nhau thng khng cho kt qu ging nhau. iu ny do cc sai s ngu nhin ca
mi quy trnh gy ra, ta khng th kim sot c hon ton tt c cc yu t nh hng n kt
qu th nghim. Do , kim sot c cc sai s ny, phi dng n khi nim chm.
chm ch ph thuc vo sai s ngu nhin v khng lin quan n gi tr thc. chm l mt
khi nim nh tnh v c biu th nh lng bng lch chun hay h s bin thin.
chm cng thp th lch chun hay h s bin thin cng ln.
73

 chm c th c phn ra thnh ba trng hp sau:

- lp li (repeatability)
- chm trung gian (intermediate precision)
- ti lp (reproducibility)
Cch xc nh
Cch 1. B tr th nghim
Tin hnh lm th nghim lp li 10 ln (t nht 6 ln) trn cng mt mu (mi ln bt u t
cn hay ong mu). Mu phn tch c th l mu chun, hoc mu trng c thm chun, tt nht l
lm trn mu th hay mu th thm chun.
Tng phng th nghim, c th b tr th nghim tnh lp li hoc chm trung gian.
Trong mt s trng hp tham gia so snh vi cc phng th nghim khc (v d trong chng trnh
th nghim thnh tho, so snh lin phng).
Nn tin hnh nng khc nhau (trung bnh, thp, cao) trong khong lm vic, mi nng
lm lp li 10 ln (t nht 6 ln). Tnh lch chun SD v lch chun tng i RSD hay h
s bin thin CV theo cc cng thc sau:

SD

( x x)

n 1

RSD% CV %

SD
100
x

Trong :
SD: lch chun
N: s ln th nghim

xi
x

: gi tr tnh c ca ln th nghim th I
: Gi tr trung bnh ca cc ln th nghim

RSD%: lch chun tng i

CV%: H s bin thin
Cch 2. Tnh ton trn cc kt qu phn tch mu thc lm.
Trong mt s trng hp vic c lng lp li c th thng qua tnh ton da trn kt
qu phn tch cc mu thc. Do vic lu gi cc kt qu phn tch c vai tr rt quan trng.

74

Da trn kt qu phn tch lm trn mu thc trong nhiu tun, t nht l 10 mu, c th
l cc nn mu khc nhau, nng khc nhau nhng phi c kt qu lm lp 2 ln .
Trng hp cc mu c nng , hm lng gn nh nhau. Tnh lch gia hai kt qu
lp ca mi mu di ri tnh lch trung bnh dtb, sau tnh lch chun s:

di
xi

Di
Dtb

RSD %

Dtb
100
1.118

Tiu ch nh gi
i chiu gi tr tnh c vi gi tr mong mun hay gi tr yu cu hoc so vi RSD%
lp li cho trong bng 7 (RSD% tnh c khng c ln hn gi tr trong bng hm lng
cht tng ng). chm thay i theo nng cht phn tch. Nng cht cng thp th kt
qu cng dao ng nhiu (khng chm) ngha l RSD cng ln.
Bng : lp ti a chp nhn ti cc nng khc nhau (theo AOAC)
TT
1
2
3
4
5
6
7
8
9
10

Hm lng %
100
10
1
0.1
0.01
0.001
0.0001
0.00001
0.000001
0.0000001

T l cht
1
10-1
10-2
10-3
10-4
10-5
10-6
10-7
10-8
10-9

n v
100%
10%
1%
0.1%
100 ppm
10 ppm
1 ppm
100 ppb
10 ppb
1 ppb

RSD (%)
1.3
1.8
2.7
3.7
5.3
7.3
11
15
21
30

Trong ni b mi phng th nghim ngoi vic tnh ton lp li, cn phi b tr th

nghim tnh c chm trung gian (mt s tc gi gi l ti lp ni b phng th
nghim) theo mt trong cc cch sau y:
- S dng cc nhn vin th nghim khc nhau;
- S dng cc thit b vi mt s c tnh khc nhau, v d cc h thng HPLC khc nhau
ca cc hng khc nhau, hoc ca cng mt hng nhng vi cc model khc nhau;
- S dng cc dung mi ha cht, thuc th c cht lng khc nhau;
75

- Khc nhau v nhit v m ca phng th nghim;

- Khc nhau cc iu kin c th ca thit b, v d thnh phn dung mi pha ng, tc
dng, pH ca pha ng...
Vic tham gia th nghim thnh tho, so snh lin phng th nghim l iu kin rt quan trng
trong nh gi phng php. Cc phng th nghim tham gia phi c kt qu th nghim lin
phng trc khi c cng nhn t ISO 17025 v duy tr cng nhn ISO 17025.

5.2.5.2. ng (trueness)
ng ca phng php l khi nim ch mc gn nhau gia gi tr trung bnh ca
kt qu th nghim v gi tr thc hoc gi tr c chp nhn l ng (). i vi a s mu
phn tch, gi tr thc khng th bit mt cch chnh xc, tuy nhin n c th c mt gi tr quy
chiu c chp nhn l ng (gi chung l gi tr ng).
Ging nh chm, ng l mt khi nim nh tnh. ng thng c din t
bng chch (bias).

Trong :

X tb
100

: chch (bias), %
Xtb: Gi tr trung bnh ca kt qu th nghim
: Gi tr thc hoc gi tr c chp nhn l ng

Cch xc nh ng
Mun xc nh ng cn phi tm c gi tr ng, c nhiu cch khc nhau xc
nh ng, bao gm vic so snh kt qu thc nghim vi kt qu thc hin bi mt phng
php i chiu hoc s dng mu bit nng (mu kim tra hoc mu chun c chng
nhn) v phng php xc nh thu hi ( tm li).
Cch 1: So snh vi phng php chun/i chiu
Phn tch mu chun hoc mu th, thc hin 10 ln bng phng php kho st v bng
mt phng php i chiu. Phng php i chiu tt nht l phng php tiu chun ca cc
t chc c uy tn, nu khng phng php i chiu l phng php qua thm nh cho kt
qu tin cy trong di o ang thc hin. Tnh ton cc kt qu trung bnh v lp li (h s bin
thin) ca hai phng php.

76

nh gi tng ng v chm ca hai phng php bng cch so snh phng sai
s2 ca hai phng php , dng tiu chun F (Fisher) v so snh hai tr gi trung bnh bng tiu
chun t (Student). Vic b tr cc th nghim phi c thc hin theo phng php tham chiu
mt cch nghim ngt v cc php o phi c tin hnh di iu kin lp li.
- So snh hai phng sai (chun F Fisher)
Chun F dng so snh lp li ca hai tp s liu hoc hai phng php khc nhau.
Vi tp s liu nh, tnh ton gi tr Ftn (F thc nghim) theo cng thc sau y v so snh vi
gi tr Fc (F tra bng).

Ftn

Trong :

Ftn

S12
1
S22

: Gi tr F thc nghim

S12 S2 2
S12 S2 2
,
: Cc phng sai ca hai phng php vi quy c
>

Nu: Ftn Fc (, k1, k2): Hai phng php c lp li ( chm) ging nhau
Nu: Ftn > Fc (, k1, k2): Hai phng php c lp li ( chm) khc nhau trong trng
hp ny nu lp li ca phng php th nghim khc vi phng php chun th cn xem
xt thm v lp li nh m t trong phn trn
Trong : Fc (, k1, k2): Gi tr F tra bng (ph lc 2) vi:
k1, k2: Bc t do (k1 = n1 -1; k2 = n2 -1)
n1, n2: S ln lm thc nghim ca hai phng php
: Mc ngha (significance level), thng thy = 0.05 (tng ng
vi tin cy (confidence level) 95%).
- So snh hai gi tr trung bnh (chun t Student)

Chun t c dng so snh xem c s khc nhau gia gi tr thc nghim v gi tr

thc hay khng; phng php ny c ng dng hoc so snh kt qu thc nghim vi gi
tr chun trong mu kim tra (xem thm cch 2) hoc so snh kt qu ca phng php phn
tch vi phng php i chiu.
Trc khi so snh hai gi tr trung bnh cn so snh hai phng sai. Vi s ln phn tch
nh hn 30, khi hai phng sai c s ng nht, tnh lch chun chung v gi tr ttn (t thc
nghim) theo cng thc sau y v so snh vi gi tr tc (t tra bng):
77

Sc 2

( n1 1) S12 ( n2 1) S2 2
n1 n2 2
x1 x2

ttn

Sc 2 (

1 1
)
n1 n2

k n1 n2 2

Trong :

ttn: Gi tr t thc nghim

tc(, k): Gi tr t tra bng mc ngha , bc t do k (xem ph lc 1)
n1, n2 : S ln th nghim ln lt ca phng php th nghim v
phng php i chiu
S12 ,S22 : Phng sai ln lt ca phng php th nghim v phng
php i chiu
x1

x2

: Gi tr trung bnh ln lt ca phng php th nghim v

phng php i chiu

Nu ttn tc(, k) : Khng c s khc nhau v kt qu ca hai phng php.
Nu ttn > tc(, k) : C s khc nhau v kt qu ca hai phng php, phng php th
nghim mc sai s h thng. Trong trng hp hai phng sai khng ng nht (khc nhau c
ngha), tnh gi tr ttn v bc t do k theo cc cng thc sau v so snh nh trn.
ttn

x1 x2
S12 S 2 2
(

)
n1 n2

S2 S 2
( 1 2 )
n1 n2

S12 S 2 2



n 2

n1
n1 1
n 2 1

Cch 2: S dng vt liu chun (Reference material)

78

Vt liu chun (cn gi l mu chun) l mu phn tch c hm lng cht phn tch
c xc nh trc v l ng. C nhiu cp vt liu chun khc nhau, trong cao nht l
CRM (certified reference material vt liu chun c chng nhn) c cung cp bi cc t
chc c uy tn trn th gii.
Cc mu CRM lun c kt qu km theo khong dao ng, do khi phn tch mu
CRM c th nh gi c ng da vo khong dao ng cho php (v d: Mu tht, ch tiu
clenbuterol l 1 ng/g 0,05 ng/g; nu kt qu phn tch c trong khong t 0,95-1,05 th t).
Nu khng c cc mu CRM c th s dng cc mu kim tra (QCQuality Control) bit nng
. Phng th nghim c th t chun b cc loi mu ny, hoc s dng cc mu thc c hm
lng bit hoc s dng cc mu lu t chng trnh so snh lin phng th nghim. Trong
trng hp khc phng th nghim c th s dng cc mu thm chun nh gi ng, ni
dung ny s c m t c th trong cch 3 di y. Nhiu t chc c uy tn nh USFDA,
EURACHEM, ICH... quy nh tnh chch (bias) xc nh ng nh sau:
x
100

Trong :

: chch (bias), %
x

: Gi tr trung bnh ca kt qu th nghim

: Gi tr thc hoc gi tr c chp nhn l ng

USFDA quy nh chch ca cc phng php xc nh d lng phi khng c ln
hn 15% v khng ln hn 20% ti LOQ. C th s dng chun t nh gi kt qu nh sau:
Phn tch mu chun lp li 10 ln (ti thiu 6 ln), tnh gi tr trung bnh v lch chun, t
tnh gi tr ttn theo cng thc sau y v so snh vi tc (p) :
ttn

Trong :

x
S2
n

ttn: Gi tr t thc nghim

tc(, k): Gi tr t tra bng vi mc ngha 0,05 (xem ph lc 1) v
bc t do k = n - 1.
: Gi tr thc hoc gi tr c chp nhn (tham chiu)
79

x : Gi tr trung bnh ca phng php th nghim

S2 : Phng sai ca phng php th nghim
n: S ln th nghim
Nu ttn tc : Khng c s khc nhau v kt qu ca gi tr trung bnh so vi gi tr tham
chiu mc ngha , tc l phng php c ng t yu cu.
Nu ttn > tc : C s khc nhau v kt qu ca phng php th nghim so vi kt qu
tham chiu mc ngha , phng php th nghim mc sai s h thng.
Cch 3: Xc nh thu hi
Cc phng php tnh ng theo cch 1 hay cch 2 u gp nhng kh khn nht
nh. Trong nhiu trng hp khng th tm hoc p dng mt phng php tiu chun so
snh kt qu, cng nh khng th d dng c c cc mu chun hoc mu chun c chng
nhn ph hp vi phng php. Vic xc nh ng do c th thc hin thng qua xc
nh thu hi (cn gi l tm li) ca phng php. Thm mt lng cht chun xc nh
vo mu th hoc mu trng, phn tch cc mu thm chun , lm lp li ti thiu bn ln bng
phng php kho st, tnh thu hi theo cng thc sau y:
- i vi mu th:
R%

Cm c Cm
100
Cc

- i vi mu trng:
R%

Trong :

Ctt
100
Cc

R%: thu hi, %

Cm+c: Nng cht phn tch trong mu thm chun
Cm: Nng cht phn tch trong mu th
Cc: Nng chun thm (l thuyt)
Ctt: Nng cht phn tch trong mu trng thm chun

Sau tnh thu hi chung l trung bnh ca thu hi cc ln lm lp li. Thm cht
chun ba mc nng l mc thp, trung bnh v cao trong khong nng lm vic. Theo
quy nh ca hi ng chu u i vi cc ch tiu an ton (cc ch tiu thuc nhm c, c quy

80

nh gii hn cho php, v d tn d hormon, khng sinh, ha cht bo v thc vt...) thm chun
vo mu trng ba mc nng ti 0,5 ln, 1 ln v 2 ln gii hn cho php (MRL).
Hi ng chu u cng quy nh i vi cc mu phn tch hng ngy (routine) cc ch
tiu thuc cng nhm (v d: ha cht bo v thc vt nhm clo hu c) cn kim sot cht
lng bng cch phn tch mu thm chun ti thiu 10% s lng cht, cc cht khc cn thay
phin kim tra vi tn sut ti a 1 nm/ln cho tng cht.
Tiu ch nh gi
Sau khi nh gi thu hi, so snh kt qu vi cc gi tr cho trong bng 8. thu hi
cc nng khc nhau c k vng khc nhau. Trong trng hp phn tch cc cht hm lng
vt c th tham kho tiu chun ca hi ng chu u (bng 9).
Bng: thu hi chp nhn cc nng khc nhau (theo AOAC)
TT
1
2
3
4
5
6
7
8
9
10

Hm lng %
100
10
1
0.1
0.01
0.001
0.0001
0.00001
0.000001
0.0000001

T l cht
1
10-1
10-2
10-3
10-4
10-5
10-6
10-7
10-8
10-9

n v
100%
10%
1%
0.1%
100 ppm
10 ppm
1 ppm
100 ppb
10 ppb
1 ppb

thu hi (%)
98-102
98-102
97-103
95-105
90-107
80-110
80-110
80-110
60-125
40-120

Bng: Quy nh v thu hi ca hi ng chu u

TT
1
2
3

Hm lng cht (g/kg)

1
>1 n <10
10

5.2.6.

n v
1 ppb
1-10 ppb
10 ppb

thu hi [%]
50-120
70-110
80-110

n nh (hay vng/ chc chn) ca phng php

n nh ca phng php l kh nng cung cp cc kt qu c chnh xc ( ng

v chm) chp nhn c di nhng iu kin c s thay i v mt s iu kin thc hin
phng php.
Ngi ta phn bit hai khi nim vng (robustness) v chc chn (rugedness) nh
sau:
81

- vng: L mc lp li cc kt qu phn tch khi c s thay i nh mt s thng

s ca phng php (method parameters). V d i vi phng php sc k lng c th k ra
cc yu t ny bao gm pH pha ng, tc dng, nhit ct, th tch tim mu, thnh phn
pha ng, bc sng pht hin...
- chc chn: L mc lp li cc kt qu phn tch khi c s thay i mt s yu t
thuc v iu kin phn tch (test conditions). Cc iu kin ny c th l ngi phn tch, thit
b phn tch, ct sc k do cc nh cung cp khc nhau, l ha cht thuc th, thi im thc
hin th nghim... Ni chung, chc chn thng lin quan n vic tnh chm trung gian
hay ti lp ni b phng th nghim.
Trong ti liu ny chng ti ni n n nh bao gm c hai yu t vng v chc
chn ca phng php.
6.2. Cch xc nh
Cho d c s khc nhau v iu kin phn tch hay khc nhau v thng s phng php,
th cng cn phi b tr cc th nghim so snh cc kt qu t c trong cc iu kin thay
i ny bng cch so snh hai phng sai (chun F) v so snh hai gi tr trung bnh (chun t)
ca hai nhm kt qu A v B. V d, gia ngi A vi ngi B, gia my A v my B, gia iu
kin A v iu kin B... Cch tin hnh so snh ging nh trong phn xc nh ng khi so
snh hai phng php nu trn. Trong trng hp c mu CRM c th b tr cc th nghim
(c s
thay i iu kin phn tch hay cc thng s ca phng php) tnh kt qu mu CRM v so
snh vi kt qu chun nh gi n nh ca phng php.

THC NGHIM

82

TI LIU THAM KHO

1.
00, P. H. H., Cy c Vit Nam. 2003, Tp 3, 431-457.
2.
Tuchinda, P. R., V.; Claeson, P.; Pongprayoon, U.; Sematong, T.; Santisuk, T.;
Taylor, C.W., Anti-inflammatory cyclohexenyl chalcone derivatives in Boesenbergia
pandurata. Phytochemistry 2002, 59 (2), 169-173.
3.
Amy, Y. L. C.; Tang, S. W.; Mohd, A. S.; Gwendoline, E. C. L.; Mawardi, R.;
Kaida, K., Characterization of flavonoid derivatives from Boesenbergia rotunda (L.).
The Malaysian Journal of Analytical Sciences 2007, 11 (1), 154-159.
4.
Kirana, C.; Jones, G. P.; Record, I. R.; McIntosh, G. H., Anticancer properties of
panduratin A isolated from Boesenbergia pandurata (Zingiberaceae). Journal of
Natural Medicines 2006, 61 (2), 131-137.
5.
Win, N. N.; Awale, S.; Esumi, H.; Tezuka, Y.; Kadota, S., Panduratins D-I, novel
secondary metabolites from rhizomes of Boesenbergia pandurata. Chemical and
Pharmaceutical Bulletin 2008, 56 (4), 491-496.
6.
Thaworn, J.; Sujin, K.; Orasa, P.; Vincent, A. P.; Vichai, R.; Pittaya, T.; Allan, H.
W., Constituents of Boesenbergia pandurata (syn. Kaempferia pandurata): Isolation,
crystal structure and synthesis of ()-boesenbergin A. Australian Journal of
Chemistry 1982, 35 (2), 351-361.
7.
Gassinee, T. K., N.; Hiroshi, S.; Makiko, T.; Mayumi, O.; Hiroshi, O.; Mitsuru, Y.;
Tadahiro, N.; Tojiro, T., Structural analysis of a novel antimutagenic compound, 4hydroxypanduratin A, and the antimutagenic activity of flavonoids in a Thai spice,
fingerroot (Boesenbergia pandurata Schult.) against mutagenic heterocyclic amines.
Journal of Agricultural and Food Chemistry 2001, 49 (6), 3046-3050.
8.
Pandji, C.; Grimm, C.; Wray, V.; Witte, L.; Proksch, P., Insecticidal constituents
from four species of the Zingiberaceae. Phytochemistry 1993, 34 (2), 415-419.
9.
Cheenpracha, S. K., C.; Ponglimanont, C.; Subhadhirasakul, S.; Tewtrakul, S.,
Anti-HIV-1 protease activity of compounds from Boesenbergia pandurata. Bioorganic
& medicinal chemistry 2006, 14 (6), 1710-1714.
10.
Win, N. N. A., S.; Esumi, H.; Tezuka, Y.; Kadota, S. , Bioactive secondary
metabolites from Boesenbergia pandurata of Myanmar and their preferential
cytotoxicity against human pancreatic cancer PANC-1 cell line in nutrient-deprived
medium. Journal Natural Product 2007, 70 (10), 1582-1587.
11.
Sukari, M. A.; Ching, A. Y. L.; Lian, G. E. C.; Rahmani, M.; Khalid, K., Cytotoxic
constituents from Boesenbergia pandurata (Roxb.) Schltr. Natural Product Sciences
2007, 13 (2), 110-113.
12.
Morikawa, T. F., K.; Ninomiya, K.; Yasuda, D.; Miyagawa, K.; Matsuda, H.;
Yochikawa, M., Medical foodstuffs. XXXIV.1) Structure of new prenylchacones and
prenylflavanones with TNF- and aminopeptidase N inhibitory activities from
Boesenbergia pandurata. Chemical and Pharmaceutical Bulletin 2008, 56 (7), 956962.
13.
Nor, A. Y.; Suffian, M. A.; Norzulaani, K., Existence of bioactive flavonoids in
rhizomes and plant cell cultures of Boesenbergia rotunda (L.) Mansf. Kulturpfl.
Australian Journal of Crop Science 2013, 7 (6), 730-734.
14.
Angkana, H.; Orasa, P.; Pittaya, T., Further studies of flavonoids of the black
rhizomes Boesenbergia pandurata. Journal of The Science Society of Thailand 1987,
13 (2), 119-122.

83

15.
Shindo, K. K., M.; Kinoshita, A.; Kobayashi, A.; Koike, Y., Analysis of
antioxidant activities contained in the Boesenbergia pandurata Schult. rhizome.
Bioscience, Biotechnology, and Biochemistry 2006, 70 (9), 2281-2284.
16.
Yoshikawa, M. M., T.; Funakoshi, K.; Ochi, M.; Pongpiriyadacha, Y.; Matsuda,
H., Medicinal foodstuffs. XXXIII.1 Gastroprotective principles from Boesenbergia
pandurata (Zingiberaceae)-absolute stereostructures of Diels-Alder type addition
prenylchalcones. Heterocycles 2008, 75 (7), 1639-1650.
17.
Tuntiwachwuttikul, P. P., O.; Reutrakul, V.; Byrne, L.T., (1'RS, 2'SR, 6'RS)-(2,6Dihydroxy-4-methoxyphenyl)-[3'-methyl-2'-(3''-methylbut-2''-enyl)-6'-phenylcyclohex-3'-enyl]methanone (Panduratin A)-a constituent of the red rhizomes of a
variety of Boesenbergia pandurata. Australian Journal of Chemistry 1984, 37 (2),
449-453.
18.
Jaipetch, T.; Reutrakul, V.; Tuntiwachwuttikul, P.; Santisuk, T., Flavonoids in the
black rhizomes of Boesenbergia pandurata. Phytochemistry 1983, 22 (2), 625-626.
19.
L. J. Jing, M. M., A. Rahmat, M. F. A. Bakar,, Phytochemicals, antioxidant
properties and anticancer investigations of the different parts of several gingers
species (Boesenbergia rotunda, Boesenbergia pulchella var attenuata and
Boesenbergia armeniaca) Journal of Medicinal Plant
Research 2010, 4 (1), 2732.
20.
Ibrahim, B. J. I., B.; Abu, S.A.; Nor, A.M.A.; Abdul, R.A.; Halijah, I. ,
Constituents of the rhizome oils of Boesenbergia pandurata (Roxb.) Schlecht from
Malaysia, Indonesia and Thailand. Flavour and Fragrance Journal 2001, 16 (2), 110
112.
21.
Sukari, M. A. S., N.W.M.; Yap, A.L.C.; Tang, S.W.; Neoh, B.K.; Rahmani, M.; Eei,
G.C.L.; Taufiq, Y.H.; Yusof, U.K., Chemical constituents variations of essential oils
from rhizomes of four Zingiberaceae species. The Malaysian Journal of Analytical
Sciences 2008, 12 (3), 638 - 644.
22.
Tewtrakul, S.; Subhadhirasakul, S.; Puripattanavong, J.; Panphadung, T., HIV-1
protease inhibitory substances from the rhizomes of Boesenbergia pandurata Holtt.
Songklanakarin Journal of Science and Technology 2003, 25 (2), 504-508.
23.
Sariga, Z.; Anuchit, P.; Niwat, K., Cytotoxic and free radical scavenging
activities of Zingiberaceous rhizomes. The Songklanakarin Journal of Science and
Technology 2005, 27 (4), 799-812.
24.
Supinya, T.; Sanan, S.; Chatchanok, K.; Chanita, P.; Sarot, C., Antiinflammatory effects of compounds from Kaempferia parviflora and Boesenbergia
pandurata. Food Chemistry 2009, 115 (2), 534-538.
25.
Lun, P., Phng Php Phn Tch Sc K V Chit Tch, chng 1. Nh xut
bn Bch Khoa H Ni, 2014.
26.
Scott, R. P. W., Liquid Chromatography Detectors. License Agreement (EULA):
2003.
27.
Yusuf, N. A. M. A., M Suffian2; Khalid, Norzulaani3, Existence of bioactive
flavonoids in rhizomes and plant cell cultures of 'Boesenbergia rotund' (L.) Mansf.
Kulturpfl. Australian Journal of Crop Science 2013, 7 ( 6 ), 730-734.
28.
Tan, B. C.; Tan, S. K.; Wong, S. M.; Ata, N.; Rahman, N. A.; Khalid, N.,
Distribution of Flavonoids and Cyclohexenyl Chalcone Derivatives in Conventional
Propagated and In Vitro-Derived Field-Grown Boesenbergia rotunda (L.) Mansf.
Evidence-based Complementary and Alternative Medicine : eCAM 2015, 2015,
451870.
29.
Ata, N.; Yusuf, N. A.; Tan, B. C.; Husaini, A.; Mohd Yusuf, Y.; Majid, N. A.; Khalid,
N., Expression profiles of flavonoid-related gene, 4 coumarate: coenzyme A ligase,
84

and optimization of culturing conditions for the selected flavonoid production in

Boesenbergia rotunda. Plant Cell, Tissue and Organ Culture (PCTOC) 2015, 123 (1),
47-55.
30.
Cheah, S. C.; Appleton, D. R.; Lee, S. T.; Lam, M. L.; Hadi, A. H.; Mustafa, M. R.,
Panduratin A inhibits the growth of A549 cells through induction of apoptosis and
inhibition of NF-kappaB translocation. Molecules 2011, 16 (3), 2583-98.
31.
Salama, S. M. A., Ahmed S. Abdulla, Mahmood A. Hassandarvish, Pouya
Bilgen, Mehmet, Protective activity of Panduratin A against Thioacetamide-induced
oxidative damage: demonstration with in vitro experiments using WRL-68 liver cell
line. BMC Complementary and Alternative Medicine 2013, 13 (1), 1-9.
32.
Yanti; Rukayadi, Y.; Lee, K. H.; Hwang, J. K., Activity of panduratin A isolated
from Kaempferia pandurata Roxb. against multi-species oral biofilms in vitro. Journal
of oral science 2009, 51 (1), 87-95.
33.
Trn Cao Sn, P. X. ., L Th Hng Ho, Nguyn Thnh Trung, Phm Gia Hu,
Phm Thanh Nh, Thm nh phng php phn tch trong ha hc v vi sinh vt.
Nh xut bn khoa hc v k thut: Vin kim nghim an ton v sinh thc phm
quc gia.

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