B GIO DC V O TO

TRNG I HC S PHM TP. H CH MINH

KHOA HA HC

KHA LUN TT NGHIP

XC NH NG THI
PARACETAMOL V IBUPROFEN
TRONG CC DC PHM BNG
PHNG PHP SC K LNG
HIU NNG CAO - HPLC
Gio vin hng dn: Th.S Nguyn Ngc Hng
Sinh vin thc hin:

Nguyn Thnh Lc

Lp: Ha 4C

Thnh ph H Ch Minh thng 5 nm 2013

MC LC
M U

.............................................................................................................. 2

Chng 1. TNG QUAN.................................................................................................... 3

1.1. Paracetamol ................................................................................................................. 3
1.1.1. Cu to

.............................................................................................................. 3

1.1.2. Tnh cht vt l v ha hc........................................................................................ 3

1.1.3. Dc tnh

.............................................................................................................. 3

1.2. Ibuprofen

.................................................................................................................. 3

1.2.1. Cu to

.............................................................................................................. 3

1.2.2. Tnh cht vt l v ha hc........................................................................................ 4

1.2.3. Dc tnh

.............................................................................................................. 4

1.3. Cc loi dc phm cha Paracetamol v Ibuprofen ................................................... 4

1.4. Tnh hnh nghin cu trong v ngoi nc .................................................................. 4
1.4.1. Phn tch Paracetamol ............................................................................................... 4
1.4.2. Phn tch Ibuprofen ................................................................................................... 5
1.4.3. Phn tch ng thi Paracetamol v Ibuprofen. ........................................................ 6
1.5. C s l thuyt v phng php sc k lng hiu nng cao ........................................ 7
1.5.1. Khi nim

.............................................................................................................. 7

1.5.2. Nguyn tc ca cc qu trnh sc k ......................................................................... 7

1.5.3. Phn loi sc k hp ph ........................................................................................... 8
1.5.4. Cc i lng c trng ca sc k ...................................................................... 9
1.5.5. H thng HPLC ....................................................................................................... 13
1.5.6. Phng php chn iu kin sc k ........................................................................ 16
1.5.7. Cc bc tin hnh sc k ....................................................................................... 19
Chng 2. THC NGHIM ............................................................................................. 21
2.1. Ha cht v dng c ................................................................................................... 21
2.1.1. Ha cht

............................................................................................................ 21

2.1.2. Dng c - Thit b.................................................................................................... 22

2.2. Ni dung nghin cu .................................................................................................. 23
2.2.1. Kho st iu kin ti u cho h thng sc k HPLC ............................................. 23
2.2.2. Xy dng phng php nh lng hn hp Paracetamol v Ibuprofen. ............... 26
2.2.3. ng dng quy trnh phn tch cc mu dc phm ................................................ 27
2.2.4. Phng php x l v nh gi kt qu .................................................................. 29

Chng 3. KT QU V THO LUN ......................................................................... 31

3.1. Kho st iu kin phn tch sc k ........................................................................... 31
3.1.1. Chn ct v th tch vng mu ................................................................................ 31
3.1.2. t bc sng cho detector ..................................................................................... 31
3.1.3.Kho st nng acid phosphoric............................................................................ 33
3.1.4. Kho st thnh phn pha ng................................................................................. 35
3.1.5. Kho st tc dng ............................................................................................... 37
3.2. Xy dng phng php nh lng ............................................................................ 39
3.2.1. Xc nh LOD v LOQ ca thit b......................................................................... 39
3.2.2. Kho st khong nng tuyn tnh ....................................................................... 39
3.2.3. Kho st lp li ca php o ............................................................................... 42
3.3. ng dng quy trnh phn tch mu tht ...................................................................... 43
3.3.1. Quy trnh chit mu ................................................................................................. 43
3.3.2. Xc nh h s thu hi ca phng php ................................................................ 44
3.3.3. nh lng Paracetamol v Ibuprofen trong mt s mu dc phm .................... 44
Chng 4. KT LUN V XUT ............................................................................ 48
4.1. Kt lun

................................................................................................................ 48

4.2. xut

................................................................................................................ 49

TI LIU THAM KHO ................................................................................................. 50

Trang 1

M U
Ngy nay vi tc pht trin nhanh chng ca khoa hc k thut, cng ngh bo
ch thuc cng khng ngng c nhng bc t ph mi c v s lng cng nh cht
lng. Cng vi xu th hi nhp th gii, Vit Nam cc ch phm thuc c bn chuyn
gim au, h st, khng vim cng c sn xut rt a dng, thnh phn khng ch
cha cc dc cht c hot tnh m cn cha nhiu loi t dc nhm tng hiu qu iu
tr. Dc phm l mt loi hng ha c bit nn khu kim nghim cht lng thuc tr
nn ht sc quan trng; iu ny em li cho ngi bnh hiu qu v an ton.
Trn th trng hin nay, cc ch phm trong v ngoi nc c tc dng iu tr
cc bnh thng thng nh cm cm, nhc u, gim au, khng vim, h st thng
c thnh phn chnh l Paracetamol, Ibuprofen v Caffein. V th nhu cu phn tch
nh tnh cng nh nh lng cc dc cht ny rt c quan tm. Vit Nam, cc
nh phn tch nghin cu ng dng nhiu phng php phn tch hin i nh trc
quang, in di mao qun, sc k lng, sc k kh xy dng quy trnh phn tch cho
mi loi dc phm khc nhau. Trn xu hng , nhiu cng trnh nghin cu nh tnh
v nh lng Paracetamol, Ibuprofen v Caffein trong cc loi dc phm c thc
hin. Trong dc in M USP30 (United State Pharmacy), phng php sc k lng
hiu nng cao (vit tt l HPLC) l phng php c quy nh cho php nh lng
ng thi Paracetamol v Ibuprofen trong cc ch phm hai thnh phn c cha ng
thi hai dc cht ny cng nh cc ch phm mt thnh phn ch cha Paracetamol
hoc Ibuprofen. Tuy nhin nhng nghin cu ng dng phng php HPLC phn tch
cc dc phm c cha hai thnh phn ny cn hn ch, cha c nghin cu su.
Cng vi nhng trang thit b hin i v c s vt cht vn c ca phng th
nghim B mn Ha Phn Tch Trng i hc S phm Tp. H Ch Minh, chng ti
quyt nh chn ti: Xc nh ng thi Paracetamol v Ibuprofen trong cc dc
phm bng phng php sc k lng hiu nng cao (HPLC) nghin cu xy dng
quy trnh phn tch cc mu dc phm c cha Paracetamol hoc Ibuprofen hoc cha
c hai thnh phn.

Trang 2

Chng 1. TNG QUAN

1.1.

Paracetamol [1]
Paracetamol (cn gi l Acetaminophen) l mt trong nhng dc cht ph bin

nht nc ta c tc dng gim au, h st nhanh .

1.1.1. Cu to
Cng thc phn t: C 8 H 9 NO 2 .
Khi lng phn t: 151,17 g/mol.
Danh php IUPAC: N-(4-hydroxyphenyl)acetamide
1.1.2. Tnh cht vt l v ha hc
T trng: 1,263 g/cm3.
Nhit nng chy: 1690C (3360F).
ha tan trong nc: 0,1 0,5 g/100 ml 200C.
Paracetamol l mt cht khng mi, c v hi ng, tinh th dng bt mu trng.
N ha tan trong cc dung mi hu c nh methanol v ethanol nhng t tan trong nc
v ether. Dung dch bo ha c pH = 5,5 6,5.
1.1.3. Dc tnh
Paracetamol l thuc gim au, h st nhng c tc dng chng vim nh aspirin.
So vi cc loi thuc khc, Paracetamol t c tc dng ph theo liu iu tr v c php
bn khng cn k n.
iu tr nhanh chng hiu qu cc cn au u, h st, cc triu chng cm cm,
au sau khi tim vaccin, au rng v khi mc rng nhng khng tr vim.
1.2.

Ibuprofen [1]
Ibuprofen l mt loi dc cht c bn trong danh mc thuc thit yu ca t chc

Y t Th gii, c bit n nh mt loi thuc chng vim dng liu thp nhng c
hiu qu cao trong iu tr.
1.2.1. Cu to
Cng thc phn t: C 13 H 18 O 2 .
Khi lng phn t: 206,28 g/mol.
Danh php IUPAC: 2-[4-(2-methylpropyl)phenyl]propanoic acid.
Trang 3

1.2.2. Tnh cht vt l v ha hc

Ibuprofen tn ti dng bt kt tinh khng mu, c mi c bit. Thc t khng
tan trong nc, tan trong ethanol, cloroform, ether v acetone. Tan c trong dung dch
kim long ca hydroxide v carbonate. V mt ha hc Ibuprofen l mt acid hu c
yu. Nhit nng chy l 760C.
Ibuprofen n nh trong mi trng khng c O 2 , ngay c nhit 105 1100C,
Ibuprofen cng bn vng t nht 4 ngy.
1.2.3. Dc tnh
Ibuprofen l thuc chng vim khng steroid, c tc dng gim au, h st, chng
vim, dng bo ch ph bin nht l vin nn.
iu tr au t nh n va v cc tnh trng vim, nh au bng kinh, nhc na
u, au hu phu, au rng, bnh c xng khp, vim khp, vim a khp dng thp,
thp khp thanh nin, bnh quanh khp, vim bao gn, m mm, bong gn, cng c.
1.3.

Cc loi dc phm cha Paracetamol v Ibuprofen

Danh mc thuc c cha Ibuprofen: Alaxan, Amfadol plus, Apo Ibuprofen,

Doaxan S, Do Parafen F, Hagifen, Hapacol au nhc, Ibuprovon, I Pan, Mofen 400,

Parofen, Protamol, Sotstop, Tatanol Extra, Vell, Ibufene Choay .
Danh mc thuc c cha Paracetamol: Aceralgin, Alaxan, Andol S, Decolgen
Forte, Panadol 500, Hapacol Extra, Efferalgan 500, Tiffy, Tatanol plus .
Cc loi thuc trn c bn rng ri trn c nc, trong cc i l thuc ty, bnh
vin v nh thuc t nhn.
1.4.

Tnh hnh nghin cu trong v ngoi nc

1.4.1. Phn tch Paracetamol

Nghin cu nh lng ng thi Paracetamol v Cafein trong ch phm bng
phng php o quang nm 2008 ca tc gi ng Thanh Thy xy dng php nh
lng ng thi hn hp hai thnh phn Paracetamol v Cafeine bng cc phng php
quang ph t ngoi nh o hm v o hm t i. ng thi p dng cc phng php
ny nh lng Paracetamol v Cafeine trong mt s bit dc thng dng trn th
trng hin nay [2].
Cng trong nm 2008, tc gi ng Qunh Chi nghin cu ti: nh lng
ng thi Paracetamol v Codein Phosphat bng phng php in di mao qun. Vi
Trang 4

nghin cu ny, tc gi xy dng phng php c th nh lng ng thi

Paracetamol v Codein phosphat. Trong iu kin in di, thi gian di chuyn ca
Codein Phosphat l 2,5 pht v ca Paracetamol l 2,9 pht, khong nng tuyn tnh
0,06 0,40 mg/ml ca Codein Phosphat v 0,19 0,120 mg/ml ca Paracetamol. Phng
php ny nhm kho st v s ph hp lp li v ng. Nhng ch phm c hm
lng hai thnh phn chnh lch nhau qu 20 ln th phn tch mu th phi tin hnh
pha long khc nhau. Phng php c p dng nh lng hm lng cc
hot cht trong vin nn Para Codein ca cng ty dc phm 3/2 [3].
Nm 2009, tc gi Deodhar MN v cng s p dng phng php sc k lng
hiu nng cao dng ct pha o (RP - HPLC) phn tch nh lng Paracetamol v
Aceclofenac vi iu kin ct pha o C 18 ( 250x4,6 mm, 10 m), pha ng l hn hp
methanol : nc c t l 70 : 30 v th tch (v/v), tc dng l 1 ml/pht vi u d
(detector) t bc sng 275 nm. Thi gian lu ca Aceclofenac v Paracetamol l 1,8
v 2,7 pht. Khong tuyn tnh l 2 50 ppm cho Aceclofenac v 5 50 ppm cho
Paracetamol. thu hi cho Aceclofenac v Paracetamol l 100,6 % v 100,7 %.
Phng php ny c nh gi c chnh xc, chn lc cao v nhanh chng nh
lng ng thi Aceclofenac v Paracetamol [4].
1.4.2. Phn tch Ibuprofen
Mt phng php thng gp trong nghin cu nh lng Ibuprofen l phng
php ph hunh quang. Nm 2001, tc gi Patricia C. Damiani cng cng s p dng
phng php ny phn tch Ibuprofen trong cc mu dc phm, kem v si r. Bc
sng kch thch 263 nm v pht x ti 288 nm. Khong nng tuyn tnh 2 73 mg/l.
Phng php ny c chn lc rt cao nn s c mt ca cc cht khc cng nh t
dc khng nh hng n kt qu phn tch, ngoi tr Chlorzoxazone trong vin nn do
Chlorzoxazone hp th mnh m trong phm vi quang ph ny. Ngoi ra cn rt nhiu
phng php khc v c bit n nhiu nht l phng php HPLC [5].
Nm 2003, S.A. Kulichenko v S.O. Fessenko nghin cu ha tan v tnh
acid base ca Ibuprofen trong h c cht hot ng b mt, sau xut mt phng
php mi c th chun Ibuprofen dng tinh khit. Gi tr pK a nh nht (4,43) v
ha tan ln nht (8,1 mg/ml) ca Ibuprofen t c khi s dng cht hot ng b mt
l dung dch tridecylpyridinium bromide 0,1 M. Php chun c thc hin vi dung
dch chun l KOH 0,05 M, ch th l xanh xylenol. Qua , nghin cu v xut iu
kin xc nh Ibuprofen v Novocaine hydrocloride [6].
Trang 5

Nm 2007, Nguyn Hu M v cng s nghin cu ng dng phng php sc k

lng hiu nng cao (HPLC) nh lng Ibuprofen trong huyt tng ch. Vi cc iu
kin sc k nh sau: ct pha o C 18 (RP-18) kch thc ct 150 x 4,6 mm, kch thc
ht l 5 m; u d (detector) t bc sng 223 nm, pha ng l hn hp acetonitril
(ACN) v dung dch m phosphat 0,02 M c pH = 6,5 (t l 30 : 70 v/v) vi tc
dng 1,2 ml/pht. Khong nng tuyn tnh ca Ibuprofen t 1 - 20 g/ml, gii hn
nh lng l 1 g/ml. Phng php ny c lp li cao, lch chun tng i
(RSD) < 3%, chnh xc, h s thu hi (R%) > 96%, hiu sut chit cao > 80%, qu
trnh chun b mu n gin, ph hp cho nghin cu dc ng hc [7].
Sau , nm 2008, tc gi Phm Thanh Huyn cng dng phng php HPLC
nh lng Ibuprofen trong huyt tng. Nhng i tng l cc bnh nhn nhi nhm
phc v cng tc theo di v iu tr bnh. iu kin tch sc k l s dng ct RP 18
(150 x 4,6 mm; 5 mm), u d (detector) t bc sng 223 nm, pha ng l hn hp
ACN v dung dch m phosphat 0,01M c pH = 6,5 (t l 60 : 40 v/v) vi tc dng
1,0 ml/pht. Khong nng tuyn tnh ca Ibuprofen t 5 - 50 g/ml, gii hn nh
lng l 5 g/ml. Phng php c lp li kh cao (RSD < 5%), chnh xc (R% >
95%), hiu sut chit cao (83%) [8].
1.4.3. Phn tch ng thi Paracetamol v Ibuprofen.
Nm 2010, tc gi Phm Thu Qu xy dng php nh lng ng thi hn hp
hai thnh phn Paracetamol v Ibuprofen bng cc phng php quang ph o hm, ly
phng php sc k lng hiu nng cao (HPLC) lm phng php i chiu. ng dng
cc phng php ny nh lng Paracetamol v Ibuprofen trong mt s l ca cc
ch phm: Alaxan, Dibulaxan v Febro [9].
Sau , tc gi M.R. Khoshayand v cng s ng dng phng php quang ph
nh lng ba thnh phnParacetamol, Ibuprofen v Caffein trong cc loi thuc, tin
hnh qut ph hp th trong khong 200 nm n 400 nm trong dung mi l hn hp
methanol : HCl 0,1 M (3 : 1) [10].
Nm 2005, bng phng php sc k lng hiu nng cao (HPLC), tc gi Thi
Duy Thn v cng s nghin cu nh lng Paracetamol, Ibuprofen v nhiu dc
cht khc trong cc mu ch phm dc ni v ngoi. Vi iu kin: ct pha o C 18
(5m, 150mm x 4,6mm), pha ng l hn hp ACN v dung dch axit phosphoric 0,1%
(60: 40 v/v), tc dng l 1,0 ml/pht vi bc sng u d (detector) t min t
ngoi l 224 nm. Thi gian lu ca Ibuprofen v Paracetamol l 2,04 v 8,22 pht. Vi
Trang 6

iu kin ny, tin hnh kho st khong nng tuyn tnh, lp li, ng, gii hn
pht hin (LOD) v gii hn nh lng (LOQ) [11].
Tc gi Prasanna Reddy Battu v MS Reddy trong nm 2009 nh lng thnh
cng Paracetamol v Ibuprofen bng phng php HPLC dng ct pha o C 18 (5 m,
150 mm x 4,6 mm), pha ng l hn hp dung mi ACN v dung dch m phosphat (pH
= 7,04) t l 60: 40 v/v, tc dng l 0,8 ml/pht vi u UV 260 nm. Thi gian
lu ca Ibuprofen, Paracetamol l 2,48 v 4,45 pht. Nghin cu trn kho st khong
nng tuyn tnh, LOD, LOQ v nh lng Paracetamol, Ibuprofen trong cc loi
dc phm bn trn th trng [12].
1.5.

C s l thuyt v phng php sc k lng hiu nng cao

HPLC l ch vit tt ca 4 ch ci u bng ting Anh ca phng php sc k

lng hiu nng cao (High Performance Liquid Chromatography), trc kia gi l sc k
lng cao p (High Pressure Liquid Chromatography).
Phng php ny ra i nm 1967 1968 trn c s pht trin v ci tin t
phng php sc k ct c in. Hin nay phng php HPLC ngy cng pht trin v
hin i ha cao nh s pht trin nhanh chng ca ngnh ch to my phn tch. Hin
nay, y l phng php c ng dng rt ln trong nhiu ngnh kim nghim nht l
kim nghim thuc. V hin ti, y l cng c t lc nht trong php phn tch nh
tnh v nh lng cc loi dc phm a thnh phn.
1.5.1. Khi nim
Sc k lng hiu nng cao 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 i bng lin
kt ho hc vi cc nhm chc hu c. Qu trnh sc k lng da trn c ch hp ph,
phn b, trao i ion hay phn loi theo kch c (ry phn t).
1.5.2. Nguyn tc ca cc qu trnh sc k
Pha tnh l mt yu t quan trng quyt nh bn cht ca qu trnh sc k v lai
sc k. Nu pha tnh l cht hp ph th ta c sc k hp ph. Nu pha tnh l cht trao
i ion th ta c sc k trao i ion. Nu pha tnh l cht lng th ta c sc k phn b hay
sc k chit. Nu pha tnh l gel th ta c sc k gel hay ry phn t. Cng vi pha tnh
ra gii cht phn tch ra khi ct, chng ta cn c mt pha ng. Nh vy nu chng ta
np mu phn tch gm hn hp cht phn tch A, B, C vo ct phn tch, kt qu l
Trang 7

cc cht A, B, C s c tch ra khi nhau sau khi i qua ct. Quyt nh hiu qu ca
s tch sc k y l tng hp cc tng tc.
Cht phn tch A +B+C
F1
Pha tnh

F2
F3

Pha ng

Hnh 1.1 S th hin s nh hng ca cc lc ra gii

Tng ca 3 tng tc ny s quyt nh cht no c ra gii ra khi ct trc
tin khi lc lu gi trn ct l nh nht (F 1 ) v ngc li. i vi mi cht, s lu gi
c quy nh bi ba lc F 1 , F 2 , F 3 . Trong F 1 v F 2 gi vai tr quyt nh, cn F 3 l
yu t nh hng khng ln. y F 1 l lc gi cht phn tch trn ct, F 2 l lc ko ca
pha ng i vi cht phn tch ra khi ct. Nh vy vi cc cht khc nhau th F 1 v F 2
l khc nhau. Kt qu l cc cht khc nhau s di chuyn trong ct vi tc khc nhau
v tch ra khi nhau khi ra khi ct nh hnh 1.2.

Hnh 1.2 Qu trnh ra gii v tch peak ca cht A v cht B.

1.5.3. Phn loi sc k hp ph
Qu trnh sc k da trn s hp ph mnh yu khc nhau ca pha tnh i vi cc
cht tan v s ra gii (phn hp ph) ca pha ng ko cht tan ra khi ct. S tch
mt hn hp ph thuc vo tnh cht ng hc ca cht hp ph. Trong loi ny c hai
kiu hp ph:
+ Sc k hp ph pha thng (NP): pha tnh phn cc, pha ng khng phn cc.
+ Sc k hp ph pha o (RP): pha tnh khng phn cc, pha ng phn cc.

Trang 8

Loi sc k ny c p dng rt rng ri, thnh cng tch cc hn hp cc cht

c tnh cht gn tng t nhau v thuc loi khng phn cc, phn cc yu hay trung
bnh nh cc vitamin, cc thuc h nhit, gim au
Ch yu hin nay chng ta s dng lai sc k hp ph pha o (RP).
Trong dc in M USP (United State Pharmacy) khi chng ta tra cu ct c gi
tr tng ng nh sau:
L 1 : RP 18

Kch thc ht tng ng t 5 - 10 m.

L7: RP 8

Kch thc ht tng ng t 5 10 m.

L3: Si 60

Kch thc ht tng ng t 5 - 10 m.

V cn mt s loi ct khc nh ct Diol Si-(CH 2 ) 2 -O-CH 2 -CH(OH)-CH 2 OH,

ct aminopropyl Si-(CH 2 ) 3 - NH 2 , ct cyanopropyl Si(CH 3 ) 2 -(CH 2 ) 3 - CN
1.5.4. Cc i lng c trng ca sc k
mV
trB
trA

trA
trB
to

Cht A

Cht B

thi gian

t 0 : thi gian lu cht.

t rA : thi gian lu cht A.

t rB : thi gian lu cht B.

t rA : thi gian lu thc cht A.

t rB : thi gian lu thc cht B.

Hnh 1.3 Sc k ca cht A v cht B.

1.5.4.1. Thi gian lu thc tR
Thi gian lu ca mt cht l thi gian tnh t khi bm mu vo ct cho n khi
cht ra khi ct t gi tr cc i. Thi gian lu ca mi cht l hng nh v cc cht
Trang 9

khc nhau th thi gian lu s khc nhau trn cng mt iu kin sc k chn. V vy
thi gian lu l i lng pht hin nh tnh cc cht.
Thi gian lu ph thuc vo cc yu t:
+ Bn cht sc k ca pha tnh.
+ Bn cht, thnh phn, tc ca pha ng.
+ Cu to v bn cht phn t ca cht tan.
+ Trong mt s trng hp thi gian lu cn ph thuc vo pH ca pha ng
Trong mt php phn tch nu t R nh qu th s tch km, cn nu t R qu ln th
peak b dong v lp li ca peak rt km, thi gian phn tch rt di ng thi ko
theo nhiu vn khc nh hao tn dung mi, ho cht, chnh xc ca php phn tch
km. thay i thi gian lu chng ta da vo cc yu t trnh by trn.
1.5.4.2. H s dung lng K
H s dung lng ca mt cht cho bit kh nng phn b ca cht trong hai
pha ng vi sc cha ct tc l t s gia lng cht tan trong pha tnh v lng cht
tan trong pha ng trong thi im cn bng.
K'=

t R t0
t0

(1.1)

Nu K nh th t R cng nh v s tch km. Nu K ln th peak b dong. Trong

thc t K t 1 - 5 l ti u.
1.5.4.3. chn lc
chn lc

cho bit hiu qu tch ca h thng sc k, khi hai cht A v B c

K A v K B khc nhau th mi c kh nng tch, mc tch biu th chn lc .

=

vi

K B'
K A'

(vi K B > K A )

cng khc 1 th kh nng tch cng r rng.

(1.2)

Hnh 1.4 nh hng ca chn lc n hiu qu tch peak ca hai cht A v B

Trang 10

1.5.4.4. S a l thuyt
S a l thuyt l i lng biu th hiu nng ca ct trong mt iu kin sc k
nht nh. Mi a l thuyt trong ct sc k ging nh l mt lp pha tnh c chiu cao
l H. Tt nhin lp ny c tnh cht ng tc l mt khu vc ca h phn tch m trong
mt cn bng nhit ng hc c thit lp gia nng trung bnh ca cht tan trong
pha tnh v pha ng.
B dy H ph thuc vo nhiu yu t:
+ ng knh v hp ph ca ht pha tnh
+ Tc v nht ( phn cc) ca pha ng
+ H s khuch tn ca cc cht trong ct.
V vy vi mt iu kin sc k xc nh th chiu cao H cng hng nh i vi
mt cht phn tch v s a l thuyt ca ct cng c xc nh.
S a l thuyt c tnh theo cng thc sau:
t
N = 5,54 R
Wo , 5

Trong ,

(1.3)

t R l thi gian lu ca cht phn tch.

W 0,5 l rng ti ca peak.

Trong thc t N nm trong khong 2500 n 5500 l va .

1.5.4.5. phn gii
phn gii l i lng biu th tch ca cc cht ra khi nhau trn mt iu
kin sc k cho. phn gii ca hai peak cnh nhau phi c tnh theo cng thc
sau:
'
'
t rB
t rA
R=2
WB + W A

(1.4)

Trong thc t nu cc peak cn i th phn gii ti thiu hai peak tch nhau
l R = 1,0. Trong php nh lng R = 1,5 l ph hp.
+ Nu R nh th cc peak cha tch hn, vic tnh ton din tch peak s khng
chnh xc, lc ny phi tm cch tng R theo ba cch sau:
* Lm thay i K bng cch thay i lc ra gii ca pha ng (thay i
phn cc nu l RP - HPLC, thay i cng ion nu l IE HPLC )
* Lm tng s a l thuyt ca ct bng cch dng ct di hn hoc ct c
kch thc nh hn.
Trang 11

* Lm tng chn lc

bng cch dng ct khc ph hp hn vi qu trnh

tch hoc thay i thnh phn pha ng .

+ Nu R ln qu th thi gian phn tch s lu, tn nhiu pha ng, nhy s
km. khc phc ta c th thay i h pha ng hay dng chng trnh gradient dung
mi. Tuy nhin trong qu trnh chy sc k nu dng chng trnh dung mi th mt s
pha ng c t l thay i s ko theo s thay i ng nn lm nh hng rt ln n
thi gian lu v din tch ca cc peak ta phn tch.
Trong thc t nn hn ch s dng chng trnh gradient dung mi m ch yu
l chng ta phi tm c h pha ng ra gii ph hp, p ng cc yu cu trong qu
trnh phn tch.
1.5.4.6. H s khng i xng
H s khng i xng T cho bit mc khng i xng ca peak trn sc k
thu c. T c tnh bng t s rng ca hai na peak ti im 1/10 chiu cao peak:

T=

a
b

(1.5)

Hnh 1.5 Cch tch h s khng i xng ca peak.

Peak dng i xng hnh Gauss trn thc t kh t c v vy phi quan tm n
h s khng i xng T.
* Khi T

2,5 th php nh lng c chp nhn.

* Khi T > 2,5 th im cui ca peak rt kh xc nh, v vy php nh lng

cn phi thay i cc iu kin sc k lm cho peak cn i hn theo cc cch sau:
+ Lm gim th tch cht tc l on ni t ct n detector.
+ Thay i thnh phn pha ng sao cho kh nng ra gii tng ln.
+ Gim bt lng mu a vo ct bng cch pha long mu hay gim
th tch tim mu.

Trang 12

1.5.5. H thng HPLC

(5)
(3)

(2)

(4)

(1)

injector

(8)

(7)

(6)

Hnh 1.6 Cc thit b trong h thng HPLC

Trong :
1 - Bnh cha dung mi pha ng.
2 - B phn kh kh.
3 - Bm cao p.
4 - B phn tim mu (tim bng syringe hay auto sampler).
5 - Ct sc k (pha tnh) ngoi mi trng hay c thit b iu nhit.
6 - u d detector (nhn tn hiu).
7 - H thng my tnh in t ci t phn mm nhn tn hiu, x l s liu v
iu khin ton b h thng.
8 Thit b in d liu.
1.5.5.1. Bnh ng dung mi
Hin ti my HPLC thng c 4 ng dung mi vo u bm cao p, cho php
chng ta s dng 4 bnh cha dung mi cng 1 ln ra gii theo t l mong mun v
tng t l dung mi ca 4 ng l 100%.
Tuy nhin theo kinh nghim th chng ta t khi s dng 4 ng dung mi cng
mt lc m chng ta ch s dng ti a l 3 hoc 2 ng cho h pha ng lun c
pha trn ng nht hn, h pha ng n gin hn qu trnh ra gii n nh .
Hin 4 ng dung mi phc v ch yu cho vic ra gii gradient dung mi theo
thi gian v cng tc xy dng tiu chun.

Trang 13

Lu : Tt c cc dung mi dng cho HPLC u phi l dung mi tinh khit v c

ghi r trn nhn l dng cho HPLC hay dung mi tinh khit phn tch.
Tt c cc ha cht dng pha mu v pha h m phi c s dng l ha cht
tinh khit phn tch v phi lc qua h thng lc 0,2 0,45 m nhm mc ch trnh lm
hng ct sc k hay nhiu ng nn, to ra cc peak tp trong qu trnh phn tch.
1.5.5.2. B phn kh kh
Mc ch ca b kh kh nhm loi tr cc bt nh cn st li trong dung mi pha
ng. Nu nh trong qu trnh phn tch m dung mi pha ng cn st cc bt kh th
mt s hin tng sau y s sy ra:
- T l pha ng ca cc ng dung mi ly khng ng s lm cho thi gian lu
ca peak thay i.
- Trong trng hp bt qu nhiu, b kh kh khng th loi tr ht c th c th
Pump s khng ht c dung mi (b e) khi p sut khng ln v my sc k s
ngng hot ng.
Trong bt c trng hp no nu trn cng cho kt qu phn tch sai.
1.5.5.3. Bm cao p
Mc ch bm pha ng vo ct thc hin qu trnh chia tch sc k. Bm phi
to c p sut cao khong 3000 - 6000 PSI hoc 250 - 500 atm (1atm = 0,98 bar) v
bm phi to dng lin tc. Lu lng bm t 0,1 n 9,999 ml/pht. Hin nay c
nhiu loi pump c p sut rt cao ln n 1200 bar.
My sc k lng ca chng ta hin nay thng c p sut ti a 412 bar. Tc
dng 0,1- 9,999 ml/pht.
Tc bm l hng nh theo thng s c ci t. Hin ti bm c 2 pistone
thay phin nhau y dung mi lin tc.
1.5.5.4. B phn tim mu
a mu vo ct phn tch theo phng php khng ngng dng chy. Vi
dung tch ca loop l 5 100 l.
C 2 cch ly mu vo trong ct: bng tim mu th cng (tim bng syringe) v
tim mu t ng (auto sampler).

Trang 14

1.5.5.5. Ct sc k
Ct cha pha tnh c coi l tri tim ca h thng sc k lng hiu nng cao.
Ct pha tnh thng thng lm bng thp khng r, chiu di ct khong 10 30
cm, ng knh trong 1- 10 mm, ht cht nhi c

= 5 - 10 m. Ngoi ra cn c mt s

trng hp c bit v kch thc v kch c ht

Vi cht nhi ct c

= 1,8 - 5 m c th dng ct ngn 3 - 10 cm v nh (ng

knh trong 1 - 4,6 mm) loi ct ny c hiu nng tch cao. Cht nhi ct ty theo lai ct
v kiu sc k (trong cc dc in USP 23, 24 c tiu chun ha cc lai ct).
Thng thng cht nhi ct l silicagel (pha thng) hoc l silicagel c
Silan ha hoc c bao mt lp mng hu c (pha o), ngoi ra ngi ta cn dng cc
loi ht khc nh: nhm oxid, polyme xp, cht trao i ion.
* i vi mt s phng php phn tch i hi phi c nhit cao hoc thp
hn nhit phng th ct c t trong thit b iu nhit. Lu : tuyt i khng nh
siu m v s lm h ct.
1.5.5.6. u d
u d (hay cn gi l detector) l b phn pht hin cc cht khi chng ra khi
ct v cho cc tn hiu ghi trn sc k c th nh tnh v nh lng. Ty theo tnh
cht ca cc cht cn phn tch m ngi ta s dng loi detector thch hp v phi tho
mn iu kin trong mt vng nng nht nh ca cht phn tch.
A = k.C
Trong :

(1.6)

A l tn hiu o c
C Nng cht phn tch
k l hng s thc nghim ca detector chn

Tn hiu ny c th l: hp th quang; cng pht x; cng in th;

dn in; dn nhit; chit sut
Trn c s ngi ta ch to cc lai detector sau:
+ Detector quang ph t ngoi t 200 n 380 nm.
+ Detector quang ph t ngoi kh kin (UV/Vis): t 190 n 900 nm.
+ Detector hunh quang pht hin cc cht hu c pht hunh quang t
nhin cng nh cc dn cht c hunh quang. y l loi detector c chn lc
cao nht.

Trang 15

+ Loi hin i hn c detector Diod Array, ELSD (detector tn x bay hi)

cc detector ny c kh nng qut chng ph nh tnh cc cht theo hp thu cc i
ca cc cht .
Ngoi ra cn c mt s loi detector khc l:
+ Detector in ha: o dng, cc ph, dn, in lng.
+ Detector chit sut vi sai: detector khc x.
+ Detector o dn nhit, hiu ng nhit.
1.5.5.7. B phn ghi tn hiu
ghi tn hiu pht hin do detector truyn sang.
+ Trong cc my th h c th s dng my ghi n gin c th v sc k
, thi gian lu, din tch ca peak, chiu cao.
+ Cc my th h mi u dng phn mm chy trn my tnh n c th lu
tt c cc thng s, ph v cc thng s ca peak nh tnh i xng, h s phn
gii... trong qu trnh phn tch ng thi x l, tnh ton cc thng s theo yu
cu ca ngi s dng nh: nng , RSD.
1.5.5.8. Thit b in d liu
Sau khi phn tch xong cc mu ta s in kt qu do phn mm tnh ton ra giy
hon thin h s.
1.5.6. Phng php chn iu kin sc k
Mun c mt kt qu tt nht ta phi tm c cc iu kin sc k tt nht cho
mt hn hp mu, cc iu kin bao gm:
Pha tnh:

- Loi pha tnh.

- Kch thc ct.

Pha ng:

- Thnh phn v t l, pH, tc dng, nhit nu l chng

trnh ra gii isocratic.

-Thnh phn v t l, pH, tc dng, nhit v chng trnh
dung mi nu l chng trnh ra gii gradient.
1.5.6.1. La chn pha tnh
Da vo cc ti liu, dc in, thnh phn v tnh cht ca cc cht c trong mu
phn tch; ta la chn ct sc k ph hp c th l ct pha thng, ct pha o hay cc
loi ct khc nhau.
Trang 16

Thng thng ngnh ta dng 2 loi ct pha thng (NP) v ct pha o (RP).
Ngoi ra ta c th dng mt s loi ct khc nh ct cyanopropyl, ct aminopropyl, ct
diol....
Ct pha thng (NP): silicagel trung tnh
Pha tnh: loi ct ny dng tch cc cht khng phn cc hay t phn
cc. Trn b mt hot ng ca n c cha cc nhm OH phn cc a nc.
Pha ng : dng cho loi ny l cc dung mi khng phn cc hay t phn
cc nh: methanol, benzen, acetonitril, chlorofom
Ct pha o (RP): silicagel alkyl ha
Pha tnh: loi ct ny dng tch cc cht khng phn cc, t phn cc,
cc cht phn cc c th to cp ion. Trn b mt hot ng cc nhm OH b alkyl
ha tc l thay th nguyn t H bng cc mch carbon thng (C 8 hay C 18 tng ng
RP-8 hay RP-18) hay cc mch carbon vng (phenyl- tng ng ct phenyl). V th n
t phn cc hay phn cc rt t.
Pha ng : Pha ng dng trong loi ny l cc dung mi c phn cc nh:
methanol, acetonitril, nc hay cc loi dung dch m, hn hp ca cc dung mi - m.
S tch ca cht nhi loi ct ny c lp li cao v n c ng dng ch yu
trong phn tch dc phm. Hin nay chng ta ch s dng loi ny l ch yu.
Hin nay pha tnh trn nn silicagel c hng trm cht khc nhau ty thuc vo
nhm th ca nguyn t H, ngoi ra cn c cc lai pha tnh trn nn oxid nhm, trn nn
cht hu c cao phn t, trn nn mch carbon .
1.5.6.2. La chn pha ng
Da vo cc ti liu, dc in, thnh phn v tnh cht ca cc cht c trong mu
phn tch; ta la chn pha ng ph hp cho qu trnh ra gii tch hon ton cc cht
c trong mu p ng y cc tiu chun ca peak trnh by ng thi phi c thi
gian phn tch ph hp nhm tit kim c dung mi, ha cht, thi gian phn tch mu,
gim thiu s hot ng ca thit b.
Pha ng c th lm thay i:
+ chn lc .
+ Thi gian lu.
+ Hiu nng tch ca ct.
+ phn gii.
+ Tnh i xng ca peak.
Trang 17

Do , trong mt pha tnh chn nu ta chn c pha ng c thnh phn ph

hp th ta s c hiu sut tch sc k tt nht i vi hn hp cc cht cn phn tch.
Chnh v vy pha ng cn c cu yu cu sau:
+ Pha ng phi tr vi pha tnh. Khng c lm cho pha tnh b bin i
ha hc ( v d gi tr pH: 2,5 < pH < 8,5).
+ Pha ng phi ha tan c cc cht phn tch th mi ra gii c
chng c bit phi ch khi thay i pha ng phi ra ct bng dung mi ph hp
khng lm ta cc cht c trong ct, hay pha ng c sn trong ct. V d: m phosphat
ra ngay bng ACN hay MeOH s b kt ta trn ct.
+ Pha ng phi bn vng theo thi gian: cng bn lu cng tt nhng t
nht l pha ng khng b phn hy trong sut thi gia phn tch mu.
+ Phi c tinh khit cao: dung mi cho HPLC, ho cht tinh khit phn
tch.
+ Phi nhanh t cn bng trong qu trnh sc k.
+ Phi ph hp vi loi detector: detector UV/Vis th dung mi khng c
hp th quang (v d: acid acetic hp th bc sng thp < 220 nm). Detector hunh
quang th dung mi khng c pht quang.
+ Phi kinh t, khng qu him v t. Trong h sc k hp ph pha o
(RP - HPLC), pha ng l dung mi phn cc: nc, ACN, MeOH, acid hay base hu c
v mt vi amin hay aminoacid
+ Ngoi cc dung mi chnh th trong thnh phn pha ng trong rt nhiu
trng hp tch RP- HPLC cn c thm hn hp m n nh pH. Cht to phc, to
cp ion to ra s ra gii tt nht.
+ Khi chn dung mi ta thng da vo lc ra gii E ca dung mi theo
bng sau:
Bng 1.1 Lc ra gii ca mt s dung mi
Dung mi

Lc ra gii E

n-Hexan

0,1

Tetra chloro carbon

1,6

Toluen

2,4

Methanol

5,11

Acetonitril

5,8

Nc ct H 2 O

10,20
Trang 18

Vic la chn iu kin sc k l cng vic ht sc cn thit trong qu trnh xy

dng chng trnh sc k. Ch khi la chn iu kin sc k tt, ph hp th chng ta
mi c th nh tnh, nh lng c cc lai thuc a thnh phn mt cch nhanh
chng v hiu qu cao.
Tt nhin phn l thuyt ni rt nhiu v cc phng php chn iu kin sc k,
tuy nhin chn c chng trnh sc k tt i hi phi c thi gian, ti liu v mt
phn kinh nghim ca nhng ngi lm sc k.
1.5.7. Cc bc tin hnh sc k
1.5.7.1. Chun b dng c v my mc
My HPLC phi c kim chng theo nh k bo m my hat ng tt,
cho kt qu phn tch c ng, lp li, tuyn tnh, t l dung mi, tc dng, nng
lng n UV ng theo yu cu thng s ca my do nh sn xut t ra.
c bit ct sc k phi c kim tra v s a l thuyt theo nh k hay khi c
nghi ng v kh nng tch v ra ng quy nh sau mi ln chy sc k:
V d: sc k pha thng NP-HPLC: ra bng methanol; khng ra bng nc.
Cn sc k pha o RP-HPLC: khi chy pha ng c cc loi mui th phi
ra nc trc cho sch.
T l ACN hay methanol : Nc l 50 : 50 v/v cho sch ht cc cht cn ng li
trong ct ng thi bo v ct khng b mc khi lu. Tuyt i trnh tnh trng ch
ra bng nc 100% sau ct mt thi gian khng s dng chc chn ct s b mc,
hng khng th dng c. Lu : nu ra ct khng tt th kt qu chy sc k s khng
th p ng c cc yu cu phn tch.
1.5.7.2. Chun b dung mi pha ng
Cc dung mi dng cho sc k l loi tinh khit dnh cho HPLC. Cc ha cht
dng phi l loi tinh khit phn tch. Pha dung mi ng, chnh xc theo ng t l
nu, n nh dung mi ng thi gian theo chuyn lun yu cu. Lc dung mi qua
mng lc 0,2 0,45 m.
1.5.7.3. Chun b mu o HPLC
Mu th: x l mu th theo ng chuyn lun, quy trnh theo nguyn tc:
- Dung mi ha tan hot cht phi ha tan trong pha ng, trong nhiu
trng hp dng dung mi pha ng ha tan mu.
Trang 19

- Phi loi b cc cht khng tan trong pha ng hoc khng ra gii c
bng cch lc hay chit .
- Phi lc v ly tm, lc mu qua mng lc 0,2 0,45 m.
- Nng mu mc va phi, khng vt qu kh nng tch ca ct. C
th gy ra nghn ct.
Mu chun:
Pha dung dch chun c thnh phn ging nh mu th trong cng dung
mi, ring v nng cc thnh phn ging nh mu th l tt nht, ngoi ra c th dng
nng khc nhng phi nm trong khong tuyn tnh kho st ca tng thnh phn.
1.5.7.4. Cch vn hnh thit b
Mi my c cch vn hnh khc nhau ty thuc vo hng sn xut, phn mm iu
khin h thng sc k HPLC. Tuy nhin cch vn hnh lun phi theo nguyn tc sau:
- Chy my vi dung mi pha ng ui ht bt kh c trong h thng
ng dn trc khi cho vo ct.
- t y cc iu kin sc k nh :
* Cu hnh my
* T l cc dung mi pha ng
* Bc sng, thnh phn mu, cc thng s ca qu trnh phn tch
yu cu.

Trang 20

Chng 2. THC NGHIM

2.1.

Ha cht v dng c

2.1.1. Ha cht
2.1.1.1. Cht chun
Bng 2.1 Danh mc cht chun do vin kim nghim thuc cung cp.
STT Tn cht chun

S l hin hnh

S l thay th

Hm lng nguyn trng (%)

Paracetamol

QT009 121911

QT009 130312

99,87%

Ibuprofen

QT026 070611

QT026 080612

99,70%

2.1.1.2. Dung mi
Bng 2.2 Danh mc dung mi tinh khit chuyn dng cho HPLC.
STT

Tn dung mi

Nh sn xut

Tiu chun

Acetonitril

Merk KGaA

Dng cho

HPLC

Merk KGaA

Dng cho

HPLC

Merk KGaA

Dng cho

HPLC

Methanol

Water

Hm lng nguyn trng (%)

99,80 %

99,80 %

99,80 %

2.1.1.3. Ha cht khc

Bng 2.3 Danh mc cc loi ha cht khc dng trong ti nghin cu.
STT

Tn ha cht

Nh sn xut

Tiu chun

Chlorofom

Merk KGaA

Dng cho

HPLC

Acid

Merk KGaA

Dng cho

phosphoric

HPLC

NaOH

Merk KGaA

Dng cho

HPLC

Trung Quc

Thng thng

Aceton

Hm lng nguyn trng (%)

99,80 %

85,00 %

99,80 %

Trang 21

2.1.2. Dng c - Thit b

2.1.2.1. Thit b
Bng 2.4 Danh mc thit b - my mc phc v ti nghin cu.
STT Loi thit b
1

Tn thit b

Bnh dng dung Pyrex 1000 ml

Hng sn xut

M s

Germany

00330341

Perkin Elmer

Series 200

Perkin Elmer

Series 200

Hamilton

80665

mi 4 knh
2

Bm cao p

B loi kh cho Vacuum Degasser

Pump

dung mi
4

Thit

tim Syringe 100 l

mu
5

Thit b np mu

Injector 20 l

Ct sc k

Brownlee

USA
Columns Perkin

RP - 18
7

Elmer 07110017

Brownlee Columns

Thit b ghi tn UV/Vis Detector

Perkin Elmer

Series 200

hiu
8

Thit b nhn tn B PC ci t phn Intel

hiu

phn mm

Total

Chrom Phn mm do Perkin

mm iu khin Workstation ver 6.1.3

Elmer cung cp

h thng
9

B lc dung mi

Glassico

India

dng giy lc
0,45
10

My

rt

chn Neuburger

KNF-Germany

N035AN.18

khng
11

Giy lc 0,45

Membrane

Filter Whatman, Japan

7141104

Sterile
12

My lc

KS130 Basic

13

My nh siu S100H Elmasonic

IKA - Germany

KS130B

Elma - Germany

101141013

m
14

Cn phn tch

Balances and Precious Sartorius

98648-012-13

Metal scales
Trang 22

15

My o quang

Lambda 25 UV/Vis Perkin Elmer

101N7062504

Spectrometor
16

My lc nc Water Pro PS

LABCONCO

091118524

siu sch
2.1.2.2. Dng c
- Bnh nh mc 1000 ml, 100ml, 50ml, 25ml, 10ml.
- Pipet: 1 ml, 2 ml, 5 ml, 10 ml.
- Bnh tam gic 250ml, cc thy tinh 100 ml, 250 ml.
- ng nh git, bnh tia, phu lc, ci s, chy s, giy lc thng.
2.2.

Ni dung nghin cu

2.2.1. Kho st iu kin ti u cho h thng sc k HPLC

2.2.1.1. Chn th tch vng mu
chnh xc, ng, lng mu cn thit np vo ct sc k khng nhng ph
thuc vo thit k ca van bm mu m cn ph thuc vo k thut np mu.
Da vo kh nng thay i cc vng mu khc nhau m c th thay i c th
tch bm mu vo ct. Tuy nhin yu t ny cng lm chn peak sc k dong ra. Nu
vng mu qu di, lng mu qu ln th hin tng dong peak xy ra cng ln gy ra
s chen ln peak trong qu trnh tch.
Lng mu c xc nh bng th tch vng cha mu m ta la chn. Vi th
tch mu nh hn th tch mu ti hn V 0 th khi bm mu vo ct tch chiu cao hay
din tch ca peak s tng tuyn tnh. n gii hn V mu = V 0 m vn tip tc tng th
tch mu th chiu cao peak sc k s khng tng na v lc peak sc k s t, dong
chn v khng sc nt. V vy vic la chn th tch cng rt quan trng.
Nu nhy phn tch, thng dng vng mu c th tch cng nh cng tt
to nn peak c sc nt cao v trnh dong peak. Trong phn tch HPLC ngi ta
thng s dng cc vng mu 10, 20, 30, 40, 50, 100 l trong vng mu 20 l thng
hay c s dng nht. Trong ti ny, nh lng Paracetamol v Ibuprofen chng
ti chn th tch vng mu l 20 l ph hp vi thit b np mu (injector) 20 l c
trang b trong phng th nghim.

Trang 23

2.2.1.2. Chn ct
Trong ti nghin cu ny, Paracetamol v Ibuprofen l nhng cht rt t phn
cc v da vo dc in M USP 23, 24 quy nh v ch tiu phn tch nh lng cc
thnh phn trong dc phm, nn ta phi chn cc loi ct RP HPLC. Trn th trng
c rt nhiu hng sn xut ct sc k pha o C 8 v C 18 . Tuy nhin ct Brownlee RP
18 ca hng Perkin Elmer c s dng rng ri trong ngnh kim nghim dc phm
nh danh ting thng hiu, cht lng ct cng nh tnh kinh t so vi cc loi ct khc.
Hin nay, phng th nghim ca ta c trang b loi ct ny v c k thut vin thng
xuyn kim tra, nh gi tnh trng. V vy, ta chn ct Brownlee RP 18, kch thc ht
5 m, 220x4,6 mm cho php nh lng ny.
2.2.1.3. Chn bc sng cho detector
Trong php phn tch ng thi, vic la chn bc sng rt quan trng v n
quyt nh ti nhy ca php phn tch. Do phng php phn tch l HPLC s dng
u d l detector UV-Vis c nh bc sng, ta cn kho st iu kin nhm chn bc
sng ti u phn tch ng thi cc cht.
Da vo ti liu tham kho v cc i hp th ca PA v IB, ta chn cc bc
sng tiu biu. Sau tin hnh kho st bc sng trn thit b HPLC ln lt ti cc
bc sng bng 220 nm, 224 nm, 250 nm, 260 nm.
2.2.1.4. Kho st bc sng trn thit b HPLC
Da vo kt qu thc nghim mc 2.2.1.3 ta tin hnh tim mu l hn hp PA
v IB nng 48 ppm vo h thng HPLC vi iu kin nh sau:
Ct tch: RP 18, 5 m, 220x4,6 mm.
Pha ng: C: Acetonitril (CH 3 CN); D: dung dch H 3 PO 4 0,01M.
Thi gian: 0 5 pht, 95%C : 5%D.
Nng mu: 4,8 ppm.
Tc dng: 1 ml/pht.
Detector: UV- Vis. t ti cc bc sng mc 2.2.1.3.
Th tch vng mu: 20 l.
2.2.1.5. Kho st nng acid phosphoric
Nng acid H 3 PO 4 nh hng trc tip ti pH ca h c th nh hng ln ti
phn cc ca dung mi nn nh hng ti kh nng tch ca h v i xng ca
Trang 24

peak sc k. kho st s nh hng ca chng, ta tin hnh chy sc k vi iu kin

sau:
Ct tch: RP 18, 5 m, 220x4,6 mm.
Pha ng: C: Acetonitril (CH 3 CN);

D: dung dch H 3 PO 4 0,001M; pH = 3,10.

E: dung dch H 3 PO 4 0,005M; pH = 2,47.
F: dung dch H 3 PO 4 0,01M; pH = 2,25.
G: dung dch H 3 PO 4 0,05M; pH = 1,81.

Thi gian: 0 5 pht, 95% C : 5% (D, E, F, G).

Nng cht phn tch: 4,8 ppm.
Tc dng: 1 ml/pht.
Detector: UV- Vis t bc sng = 224 nm.
Th tch vng mu: 20 l.
2.2.1.6. Kho st thnh phn pha ng v Ki
T l thnh phn dung mi to ra pha ng c nh hng ln n qu trnh ra gii
cc cht mu ra khi ct. Trong phn tch HPLC, khi nim lc ra gii l c trng cho
qu trnh sc k. Khi t l thnh phn pha ng thay i th lc ra gii ca dung mi
pha ng thay i, ngha l lm thay i thi gian lu ca cc cht phn tch qua lm
thay i h s lu ca cht phn tch . V vy c c t l thnh phn pha ng
ph hp cn tin hnh kho st h sc k vi t l thnh phn pha ng khc nhau vi
cc iu kin sc k nh nhau:
Ct tch: RP 18, 5 m, 220x4,6 mm.
Pha ng: C: Acetonitril (CH 3 CN); D: dung dch H 3 PO 4 0,010 M.
Thi gian: 0 5 pht,

60%C : 40%D

70%C : 30%D

80%C : 20%D

90%C : 10%D

95%C : 05%D

96%C : 04%D

97%C : 03%D

98%C : 02%D

99%C : 01%D.
Nng cht phn tch: 48 ppm.
Tc dng: 1 ml/pht.
Detector: UV- Vis t bc sng = 224 nm.
Th tch vng mu: 20 l

Trang 25

2.2.1.7. Kho st tc dng

Tc pha ng cng l yu t nh hng ti qu trnh sc k v n nh hng
n qu trnh thit lp cn bng ca cht tan gia pha tnh v pha ng. Tc dng qu
nh s gy ra hin tng dong peak, thi gian ra gii lu hn. Tuy nhin nu tc
dng qu ln c th lm cho cc cht trong hn hp khng tch khi nhau hon ton,
ngha l gy ra hin tng chng cho peak ln nhau. V vy cn phi kho st tm ra
tc dng ph hp vi h phn tch.
Ct tch: RP 18, 5 m, 220x4,6 mm.
Pha ng: C: Acetonitril (CH 3 CN); D: dung dch H 3 PO 4 0,01M.
Thi gian: 0 5 pht,

97%C : 3%D.

Nng cht phn tch: 48 ppm.

Tc dng: 0,6 ml/pht; 0,7 ml/pht; 0,8 ml/pht; 0,9 ml/pht; 1 ml/pht.
Detector: UV- Vis t bc sng = 224 nm.
Th tch vng mu: 20 l.
2.2.2. Xy dng phng php nh lng hn hp Paracetamol v Ibuprofen.
2.2.2.1. Xc nh gii hn pht hin LOD v gii hn nh lng LOQ
Trong mt quy trnh phn tch bt k, gii hn pht hin (LOD) v gii hn nh
lng (LOQ) l hai thng s quan trng.
Gii hn pht hin l gi tr nng thp nht ca cht phn tch m h thng phn
tch cn cho tn hiu phn tch c ngha so vi tn hiu mu trng hay tn hiu ca ng
nn.
Gii hn nh lng c xem l nng thp nht ca cht phn tch m h
thng phn tch nh lng c vi tn hiu phn tch c ngha nh lng so vi tn
hiu mu trng hay tn hiu nn. Thng thng LOQ c tnh nh sau:
xc nh LOD v LOQ ca thit b, ta tin hnh nh sau: pha long t 5 7 ln
mu chun cha hn hp PA v IB nng 9,6 ppm v chun b mt mu trng (cha
dung mi pha ng). Sau tim vo thit b HPLC v ghi kt qu. Sc k no th
hin chiu cao peak khong gp 3 ln ng nn th nng ca mu chun chnh l
gii hn pht hin LOD ca thit b.
Gii hn nh lng c suy ra t cng thc: LOQ = 10/3 LOD

(2.1)

Trang 26

2.2.2.2. Xc nh khong nng tuyn tnh

Khong nng tuyn tnh l mt thng s quan trng ca quy trnh phn tch.
Mt cht ch c th nh lng tt theo phng php ng chun hay thm chun khi
nng ca cht phn tch nm trong khong tuyn tnh.
kho st khong tuyn tnh gia nng ca Paracetamol, Ibuprofen v din
tch peak sc k, ta ln lt pha cc dung dch chun c nng t 4,8 ppm n 480 ppm
trong dung mi pha ng. Sau tim vo h thng HPLC vi iu kin ti u c
kho st mc 1.2.2, ghi gi tr v dng phn mm Microsoft Excel 2003 xy dng
ng hi quy.
Cc thng tin v ng chun c trnh by trong phn ph lc.
2.2.2.3. Kho st lp li ca php o
Mt phng php phn tch tt ngoi yu cu v ng ca phng php, ngi
ta cn ch n lp li ca phng php. lp li ca h thng sc k c kho
st bng cch tim lp 6 ln cng mt mu chun hn hp Paracetamol v Ibuprofen (c
nng nm trong khong tuyn tnh) vo h thng sc k vi iu kin ti u c
kho st mc 1.2.2. Kt qu c nh gi thng qua lch chun (SD), lch
chun tng i (RSD) ca din tch peak sc k S peak v thi gian lu t R .
2.2.3. ng dng quy trnh phn tch cc mu dc phm
2.2.3.1. Kho st quy trnh chit cht:
Cn 10 vin mu thuc, ghi li kt qu cn.
Nghin mn mu bng chn s sau cn chnh xc 0,2504 gam mu.
Cho 0,2504 g mu vo bnh bnh tam gic c cha 30 ml dung dch pha ng. Lc
trong 15 pht. Sau lc tch phn cn khng tan v phn dung dch.
Tng t lp li cc bc nh s sau:

Trang 27

Hnh 2. 1 Quy trnh kho st phng php chit mu

C thc hin cho n khi cht chit hon ton. Thu ly cc dch lc c cha
cht v dch lc khng cha cht (lm mu trng).
2.2.3.2. Xc nh h s thu hi ca phng php
Nhm nh gi v kim tra ng ca phng php, ta tin hnh xc nh h s
thu hi bng cch sau:
Tim lp 3 ln dung dch cha ng thi PA v IB nng C 01 , C 02 xc nh (C 01
v C 02 phi nm trong khong tuyn tnh). Ghi s liu, da vo ng chun ca PA v
IB tnh nng C 1 , C 2 ca PA v IB. Sau suy ra h s thu hi theo cng thc sau:
R(%) =

Ci
.100%
C oi

(2.2)

2.2.3.3. Phn tch nh lng mt s mu dc phm

Trn th trng hin nay c rt nhiu loi dc phm c cha PA, IB v ng thi
PA, IB trong cng mt ch phm. Da vo phng php ny ta c th nh lng PA, IB
Trang 28

hay ng thi PA v IB trong cng mt ch phm. Tuy nhin, ta khng th trnh khi s
nh hng ca cc cht khc trong cc loi dc phm a thnh phn. V vy, ta nn
chn cc loi dc phm ch cha mt thnh phn PA hoc IB hay ng thi hai thnh
phn PA v IB. Ngoi ra, ta cng c th nh lng PA v IB trong mt s dc phm a
thnh phn m dc in cho php hoc c cc ti liu chng minh cht l khng nh
hng n php phn tch.
Bng 2.5 Thng tin v cc mu thuc c thu thp
Tn mu

Tn thuc

Thnh phn chnh

Nh sn xut s l

HSD

Paracetamol 325 mg

United Pharma Vit

23/05/2014

Ibuprofen 200 mg

Nam - 118691

Paracetamol 325 mg

United Pharma Vit

Ibuprofen 200 mg

Nam - 118901

trong mt vin thuc

M 01

M 11

Alaxan

Alaxan

24/10/2014

Tin hnh x l mu thuc theo quy trnh thc nghim suy ra t mc 2.2.3.1. Thu
ly ton b dch chit, lc qua mng lc 0,45 m, sau cho ton b dch lc vo bnh
nh mc 250 ml. nh mc n vch bng dung mi pha ng v pha long nh sau:
Bng 2.6 Cch pha long mu trc khi tim vo thit b HPLC.
Stt Mu thuc Alaxan
1

M 01

Cch pha long

Rt 2,5 ml mu cho vo bnh 25 ml, nh mc n vch bng
dung mi pha ng.

M 11

Rt 7,5 ml mu cho vo bnh 25 ml, nh mc n vch bng

dung mi pha ng.

Tin hnh tim lp 3 ln cc mu M 01 , M 11 vo h thng HPLC v ghi kt qu.

Da vo phng trnh hi quy tnh nng ca cht phn tch v h s thu hi.
2.2.4. Phng php x l v nh gi kt qu
Theo l thuyt sc k lng, trong mt iu kin sc k xc nh chn, th thi
gian lu ca cht l i lng c trng nh tnh (pht hin) cc cht. Cn chiu cao
v din tch peak sc k c lin quan cht ch n nng ca cht. Trong mt vng
nng nht nh v khng ln, th chng ta c mi quan h tuyn tnh nh sau:
H i = k 1 .C i = f(C)

(2.3)

S i = k 2 .C i = f(C)

(2.4)
Trang 29

trong :
H i v S i l chiu cao v din tch ca peak sc k ca cu t i.
C i l nng ca cu t i vi thi gian lu t Ri .
k 1 , k 2 l cc hng s thc nghim ph thuc vo cc iu kin sc k cng nh
bn cht pha tnh.
Da trn (2.3) v (2.4) ta c th xc nh nng cc cht phn tch theo phng
php ng chun hay thm chun.
Cc s liu thc nghim c x l bng phng php thng k ton hc vi cc
c trng sau:
Gi tr trung bnh:

x=

1 n
xi
n i =1

(2.5)

(x
n

lch chun:

SD =

lch chun tng i:

RSD(%) =

i =1

n 1
s
.100
x

(2.6)

(2.7)

Trang 30

Chng 3. KT QU V THO LUN

3.1.

Kho st iu kin phn tch sc k

Sau khi tin hnh cc th nghim kho st theo mc 2.2.1, ta thu c kt qu nh sau:
3.1.1. Chn ct v th tch vng mu
Da vo cc ti liu tham kho trong mc 1.4.3 v trang thit b ca phng th
nghim cho php ta chn ct v th tch vng mu nh sau:
Ct sc k pha o RP 18, kch thc 220x4,4 mm, kch thc ht 5 m.
Th tch vng mu: injector 20 l.
3.1.2. t bc sng cho detector
Bc sng ca detector l mt yu t quan trng, nh hng n nhy ca
phng php. V th ta phi nghin cu, kho st sao cho tn hiu ca cht phn tch ti
bc sng l tt nht.

Hnh 3.1 Ph hp th ca PA v IB trong dung mi NaOH 0,1M [13].

Hnh 3.2 Ph hp th ca PA, IB v CA trong dung mi MeOH : HCl 0,1M (3:1 v/v) [11].

Trang 31

Nhn xt: PA hp thu cc i trong khong bc sng 250 260 nm. IB hp th

cc i trong khong 220 230 nm.
Tin hnh kho st bc sng trn detector t ti cc bc sng 220 nm, 224 nm,
250 nm, 260 nm. Tim hn hp cha PA v IB nng 48 ppm trong dung mi pha
ng vo h thng HPLC vi iu kin nh mc 2.2.1.4.
Bng 3.1 S ph thuc ca chiu cao peak sc k vo bc sng ca detector
Bc sng detector

Chiu cao peak

PA
325
1510
220
112

220
224
250
260

IB
265
1230
217
102

mV

mV

Thi gian(pht)

Thi gian(pht)

(a)

(b)

mV

mV

Thi gian(pht)

Thi gian(pht)

(c)

(d)

Hnh 3.3 Sc k th hin chiu cao peak sc k ti cc bc sng

(a) 220 nm, (b) 224 nm, (c) 250 nm, (d) 270 nm.
Trang 32

1600
1400
1200
1000
800
600
400
200
0

PA
IB

215

220

225

230

235

240

245

250

255

260

265

Hnh 3.4 S ph thuc ca chiu cao peak sc k vo bc sng ca detector.

Nh vy, t cc kt qu trn ta nhn thy: tc v thnh phn pha ng c th
lm chuyn dch cc i hp th ca PA v IB. tch c ng thi PA v IB, ta chn
bc sng 224 nm cho cc nghin cu tip theo.
3.1.3. Kho st nng acid phosphoric
Trong nghin cu ny, ta chn acid phosphoric v PA v IB theo l thuyt u c
tnh acid yu. Nu chn mi trng trung tnh hoc base th PA v IB c th tn ti
dng mui v kt tinh trn ct khi gp MeOH hay ACN.
Tuy nhin ta cng cn phi kim sot nng ca acid H 3 PO 4 . Nu pH qu thp,
vt qu gii hn ca ct tch s lm bin i pha tnh.
Tin hnh kho st nng acid H 3 PO 4 theo mc 2.2.1.5. Kt qu c th hin
trong bng 3.2 v hnh 3.5 nh sau:
Bng 3.2 S ph thuc h s lu K i vo nng acid H 3 PO 4 :
Nng acid

pH

H 3 PO 4 (M)

H s lu K i
PA

IB

0,001

3,10

2,47

3,23

0,005

2,47

2,44

3,24

0,01

2,25

2,41

3,19

0,05

1,81

2,40

3,23

Trang 33

3.3
3.2
3.1
3
2.9
2.8
2.7
2.6
2.5
2.4
2.3

PA
IB

0.01

0.02

0.03

0.04

0.05

0.06

Hnh 3.5 S ph thuc thi gian lu vo nng acid H 3 PO 4 .

mV

tIB = 3,95
tPA = 3,23

t0 = 0,93

Thi gian(pht)

Hnh 3.6 Sc k ca hn hp PA v IB (4,8 ppm) vi nng acid H 3 PO 4 0,001M.

Da vo kt qu phn tch c, ta chn nng acid H 3 PO 4 l 0,01 M cho cc
nghin cu tip theo v ti nng ny, pH nm trong gii hn cho php ca ct, peak
sc k r nt, cn i, t tn thi gian phn tch v dung mi pha ng.

Trang 34

3.1.4. Kho st thnh phn pha ng

Thnh phn pha ng nh hng trc tip n lc ra gii cc cht phn tch, nh
hng n thi gian lu v h s tch ca hai cht. Theo dc in M USP 23, 24 v
cc ti liu tham tham kho mc 1.4 th dung mi pha ng thng l hn hp ACN v
mt acid yu nh: acid acetic, acid formic, acid phosphoric Trong ti ny, ta chn
acid phosphoric v n l acid v c, khng hp th bc sng 224 nm.
Tin hnh kho st thnh phn pha ng nh mc 2.2.1.6. Kt qu c th hin
trong hnh 3.7 nh sau:
mV

mV

Thi gian(pht)

Thi gian(pht)

mV

mV

Thi gian(pht)

Thi gian(pht)

(3)

(4)

Trang 35

mV

mV

Thi gian(pht)

Thi gian(pht)

(5)

(6)
mV

mV

Thi gian(pht)

Thi gian(pht)

(7)

(8)

mV

Hnh 3.7. Sc k ca hn hp PA v IB
vi pha ng l:
(1) ACN/ H3PO4 0.01M (60:40 v/v).
(2) ACN/ H3PO4 0.01M (70:30 v/v).
(3) ACN/ H3PO4 0.01M (80:20 v/v).
(4) ACN/ H3PO4 0.01M (90:10 v/v).
(5) ACN/ H3PO4 0.01M (95:5 v/v).
(6) ACN/ H3PO4 0.01M (96:4 v/v).
(7) ACN/ H3PO4 0.01M (97:3 v/v).
Thi gian(pht)

(9)

(8) ACN/ H3PO4 0.01M (98:2 v/v).

(9) ACN/ H3PO4 0.01M (99:1 v/v).

Da vo hnh 3.7, ta thy rng thnh phn pha ng nh hng ln n thi gian
lu, h s lu, h s tch v h s i xng ca peak.
Trang 36

T l ACN cng ln th thi gian lu cng ngn, h s tch cng nh, peak p,
cn i, hin tng ko ui gim dn. Tuy nhin, khi t l ACN ln n 98 99 % th
h s tch qu nh, hai peak khng tch ri nhau. V vy ta chn thnh phn pha ng l
hn hp dung mi ACN v dung dch H 3 PO 4 0,01M (97:3 v/v) cho nhng nghin cu
tip theo.
3.1.5. Kho st tc dng
Tc pha ng cng l yu t nh hng ti qu trnh sc k v n nh hng
n qu trnh thit lp cn bng ca cht tan gia pha tnh v pha ng. Tc dng qu
nh s gy ra hin tng dong peak, thi gian ra gii lu hn. Tuy nhin nu tc
dng qu ln c th lm cho cc cht trong hn hp khng tch khi nhau hon ton,
ngha l gy ra hin tng chng cho peak ln nhau. V vy cn phi kho st tm ra
tc dng ph hp vi h phn tch.
Tin hnh kho st theo mc 2.2.1.7. Kt qu kho st c th hin hnh 3.8
T kt qu trong hnh 3.8 ta thy rng, tc dng nh hng ln n thi gian
lu, h s tch. Tc dng t 0,6 1,0 ml/pht cho thy thi gian lu gim dn, peak
r p, tch nhau hon ton, h s tch gim dn. Tc dng ln s lm hao tn dung
mi v tng p sut ct tch, nhng tc qu nh s ko di thi gian phn tch v lm
dong peak gy ra hin tng ko ui. Do , ta chn tc dng l 0,7 ml/pht tin
hnh nh lng PA v IB.
Tm li, iu kin ti u c xut nh sau:
Ct tch: RP 18, 5 m, 220x4,6 mm.
Pha ng: Acetonitril (CH 3 CN) : dung dch H 3 PO 4 0,01M (97:3 v/v).
Thi gian: 0 4,5 pht.
Tc dng: 0,7 ml/pht.
Detector: UV- Vis t bc sng = 224 nm.
Th tch vng mu: 20 l.

Trang 37

mV

mV

Thi gian(pht)

Thi gian(pht)

(1)

(2)

mV

mV

Thi gian(pht)

(3)

Thi gian(pht)

(4)

mV

Hnh 3.8 Sc k hn hp PA v IB vi tc
dng thay i
(1) tc dng = 1,0 ml/pht
(2) tc dng = 0,9 ml/pht
(3) tc dng = 0,8 ml/pht
(4) tc dng = 0,7 ml/pht
Thi gian(pht)

(5) tc dng = 0,6 ml/pht.

(5)

Trang 38

3.2.

Xy dng phng php nh lng

3.2.1. Xc nh LOD v LOQ ca thit b

Trong mt quy trnh phn tch bt k, gii hn pht hin (LOD) v gii hn nh
lng (LOQ) l hai thng s quan trng.
xc nh LOD v LOQ ca thit b, ta tin hnh nh sau: chun b mt mu
trng lm nn, pha long lin tip 2 n 5 ln mu chun cha hn hp PA v IB. Sau
tim vo thit b HPLC. n khi no chiu cao peak = 3 ln chiu cao ng nn th ly
l LOD.
mV

mV

(a)

(b)

Thi gian(pht)

Thi gian(pht)

Hnh 3.9 Mu hn hp PA v IB nng 1,2 ppm (a) v mu trng (b).

Kt qu: khi nng PA v IB l 1,2 ppm th chiu cao = 3 ln ng nn. Do :
LOD ca PA l 1,2 ppm
LOD ca IB l 1,2 ppm
T suy ra:

LOQ ca PA = 10/3 LOD = 4 ppm.

LOQ ca IB = 10/3LOD = 4 ppm.

3.2.2. Kho st khong nng tuyn tnh

Khong nng tuyn tnh l mt thng s quan trng ca quy trnh phn tch.
Mt cht ch c th nh lng tt theo phng php ng chun hay thm chun khi
nng ca cht phn tch nm trong khong tuyn tnh.
kho st khong tuyn tnh gia nng ca Paracetamol, Ibuprofen v din
tch peak sc k, ta ln lt pha cc dung dch chun c nng t 4,8 ppm n 360 ppm
trong dung mi pha ng.
Trang 39

Bng 3.3 Cch pha long dung dch mu trc khi tim vo h thng HPLC
Th tch (ml) ht t dung
dch chun 1200 ppm
0,1
0,2
0,5
1,0
2,0
5,0
7,5

H s pha long
0,1/25
0,2/25
0,5/25
1,0/25
2,0/25
5,0/25
7,5/25

Nng ca dung dch

sau khi pha long
4,8
9,6
28,8
57,6
115,2
240
360

Sau tim vo h thng HPLC vi iu kin ti u c kho st mc 1.2.2,

ghi gi tr v dng phn mm Microsoft Excel 2003 xy dng ng hi quy.
Bng 3.4 Din tch peak ng vi tng nng ca PA trong dy chun.
Paracetamol
C (ppm)

S peak

4,8

1624,11

9,6

111005,47

28,8

397690,90

57,6

914222,87

115,2

1945623,06

240

4042599,58

360

5975959,25

Bng 3.5 Din tch peak ng vi tng nng ca IB trong dy chun.

Ibuprofen
C (ppm)

S peak

4,8

224205,66

9,6

320008,04

28,8

841851,68

57,6

1528423,76

115,2

2894882,44

240

6028463,40

360

9161289,52
Trang 40

ng chun cua Paracetamol

7000000,00

6000000,00

5000000,00

mAU

4000000,00
y = 16879x - 54957
2
R = 0,9997

3000000,00

2000000,00

1000000,00

0,00
0,00

100,00

200,00

300,00

400,00

Nng (ppm)

Hnh 3.10 th biu s ph thuc din tch peak vo nng ca Paracetamol.

ng chun cua Ibuprofen
10000000,00
9000000,00
8000000,00
7000000,00
y = 25061x + 78528

mAu

6000000,00

R = 0,9998

5000000,00
4000000,00
3000000,00
2000000,00
1000000,00
0,00
0,00

50,00

100,00 150,00 200,00 250,00 300,00 350,00 400,00

Nng (ppm)

Hnh 3.11 th biu din s ph thuc din tch peak vo nng ca IB

Trang 41

Da vo kt qu kho st, ta c cc phng trnh hi quy sau c biu din trong

hnh 3.10, 3.11 v bng 3.6.
Bng 3.6 Phng trnh hi quy ca PA v IB
Cht phn tch

Phng trnh hi quy

H s tng quan

Khong nng

R2

tuyn tnh (ppm)

Paracetamol

Y = 16879x 54957

0,9997

4,8 360

Ibuprofen

Y = 25061x + 78528

0,9998

4,8 - 360

Nhn xt: qua cc kt qu phn tch v thng k ta thy, t l din tch peak sc k
v nng cc acid bo ph thuc tuyn tnh vi nhau mt cch cht ch vi h s tng
quan cao, R = 0,9997. Khong nng tuyn tnh rng i vi c hai cht PA v IB t
4,8 360 ppm. Do , ta c th s dng cc phng php ng chun hay thm chun
nh lng PA v IB trong mu tht khong tuyn tnh kho st.
3.2.3. Kho st lp li ca php o
Mt phng php phn tch tt ngoi yu cu v ng ca phng php, ngi
ta cn ch n lp li ca phng php. lp li ca h thng sc k c kho
st bng cch tim lp 7 ln cng mt mu chun hn hp PA v IB vo h thng HPLC
vi iu kin ti u mc 1.2.2. Kt qu c nh gi thng qua lch chun (SD),
lch chun tng i (RSD) ca din tch peak sc k S peak v thi gian lu t R .
Bng 3.7 lp li ca h thng HPLC vi mu PA
Thi gian lu ca PA

Th t

Thi gian lu

Cc thng s thng k

Din tch peak ca PA

Din tch

Cc thng s thng k

2,25

Gi tr trung bnh

274172,44

Gi tr trung bnh

2,26

X tb = 2,27

274378,18

X tb = 275718,71

2,27

lch chun:

278549,62

lch chun:

2,27

SD = 0,011

278947,38

SD = 2206,77

2,27

lch chun tng i:

275137,04

lch chun tng i:

2,28

RSD(%) = 0,47 %

275626,77

RSD(%) = 0,80 %

2,28

273219,56

Trang 42

Bng 3.8 lp li ca h thng HPLC vi mu IB

Thi gian lu ca IB

Th t

Thi gian lu

Din tch peak ca IB

Cc thng s thng k

Din tch

Cc thng s thng k

3,89

Gi tr trung bnh

575941,83

Gi tr trung bnh

3,90

X tb = 3,90

576812,77

X tb = 577933,34

3,89

Sai s ngu nhin:

580467,34

Sai s ngu nghin:

3,90

SD = 0,011

583875,91

SD = 3263,80.

3,91

lch chun tng i:

577994,18

lch chun tng i:

3,91

RSD(%) = 0,29 %

576278,13

RSD(%) = 0,56 %

3,92

574163,22

Theo kt qu kho st ta thy h thng sc k lng c lp li tt. i vi php

nh lng, thng s din tch peak S peak l 0,80 % v 0,56 % ng vi PA v IB. Quy
trnh phn tch n nh v c th p dng phn tch mu tht.
3.3.

ng dng quy trnh phn tch mu tht

3.3.1. Quy trnh chit mu

Mu thuc Alaxan c thu thp t cc hiu thuc v, ghi li cc thng tn v bao
b, ngy sn xut, hn s dng, s ng kim v thnh phn nh trong bng 2.5.
Sau khi thc hin quy trnh kho st phng php chit mu, kt qu thu c th
hin trong bng 3.9 v hnh 3.12:
Bng 3.9 Din tch peak vi s ln chit tch PA v IB trong mu tht.
S ln chit i

Mu Alaxan
S peak PA

S peak IB

2262527,5

339689,29

109617,87

24555,11

46703,42

14352,24

16639,31

11064,97

5589,70

4183,39

4845,69

1946,18

Trang 43

(b)

(a)

Hnh 3.12 S ph thuc ca din tch peak vo s ln chit PA (a) v IB (b).

Nhn xt, nh vy sau 7 ln lc v chit xem nh ta tch hon ton PA v IB ra
khi mu ban u l 250 mg thuc Alaxan. Da vo y, ta c th p dng quy trnh ny
chit tch PA v IB cho php nh lng.
3.3.2. Xc nh h s thu hi ca phng php
xc nh ng ca phng php, ta cn xc nh h s thu hi.
Tim lp 3 ln dung dch cha ng thi PA v IB nng 120 ppm. Ghi s liu,
da vo ng chun ca PA v IB trong bng 3.7, tnh nng ca PA v IB. Sau
tnh h s thu hi theo cng thc 2.2.
Kt qu ca php o c th hin trong bng 3.10:
Bng 3.10 H s thu hi ca phng php trn mu chun PA v IB
Mu

PA

IB

Nng C x (ppm)

115,23

113,38

Nng C 0 (ppm)

120

120

H s thu hi R%

96,03 %

94,48 %

3.3.3. nh lng PA v IB trong mt s mu dc phm

Vi phng php nh lng PA v IB xy dng, ta tin hnh phn tch 2 mu:
Alaxan c s l v ngy sn xut khc nhau.
Khi lng 10 vin thuc ly t mu M 01 : 7,2061 gam.
Khi lng 10 vin thuc ly t mu M 11 : 7,2045 gam.
Suy ra khi lng trong 1 vin thuc ly t mu M 01 : 720,61 mg.
khi lng trong 1 vin thuc ly t mu M 11 : 720,45 mg.
Tin thnh cn ly mu: Khi lng mu M 01 = 250,9 mg.
Khi lng mu M 11 = 251,1 mg
Trang 44

Quy trnh x l mu v chit cht thc hin theo mc 2.2.3.1 v pha ch theo bng
2.6. Da vo phng trnh hi quy t bng 3.6 xc nh hm lng PA v IB trong
mu. Kt qu phn tch c th hin trong bng v hnh sau:

Hnh 3.13 Sc k ca mu M 01 .

Hnh 3.14 Sc k mu M 11 .
Trang 45

Bng 3.11 Kt qu phn tch mu dc phm Alaxan M 01 v M 11 .

Mu S peak PA

S peak IB

C PA (ppm)

M 01

708161,69

673225,53

45,21

C tb =

23,73

C tb =

709626,28

679240,17

45,30

45,2 0,30

23,97

23,9 0,4

705685,92

680242,61

45,06

M 11

C IB (ppm)

24,01

2266033,80 1906727,95 137,51

C tb =

72,95

C tb =

2290019,04 1898207,21 138,93

137,3 3,4

72,61

72,9 0,5

2231189,70 1905224,29 135,44

72,89

T nng PA v IB tm c trong mu, ta quy i sang hm lng ca chng

c trong 100 mg mu nh sau:
m cpt = C x .V.F
trong :

m cpt l khi lng cht phn tch c trong 0,2509 gam mu (i vi

mu M 01 ) v 0,2511 gam mu (i vi mu M 11 ).
C x l nng cht phn tch tnh theo phng trnh hi quy.
V: th tch pha long.
F: h s pha long (10-3).
Suy ra: m cpt = C x . 250.
m cpt = C x . 250.

25
.10 3 = 2,5C x i vi mu M 01 .
2,5

25
.10 3 = 0,83C x i vi mu M 11 .
7,5

Nh vy, theo quy trnh phn tch trn ta c hm lng PA v IB trong mu

dc phm Alaxan c tm tt nh sau:
Bng 3.12 Khi lng PA v IB trong mu M 01 v M 11 .
Mu Khi lng
cn (mg)

Khi lng PA

Khi lng IB

(mg)

(mg)

Khi lng PA Khi lng IB

trong 1 vin

trong 1 vin

thuc

thuc

M 01

250,9

112,9 0,7

59,8 0,9

324,5 2,2

171,6 2,7

M 11

251,1

113,9 2,8

60,5 0,4

326,9 8,0

173,5 1,2

Trang 46

Bng 3.13 Hm lng PA v IB trong mu dc phm Alaxan

Mu

H s thu hi (%)
PA

IB

M 01

99,8 0,7

85,8 1,4

M 11

100,6 2,5

86,8 0,6

Da vo kt qu phn tch hm lng PA v IB trn ta nhn xt nh sau

Hm lng PA v IB trong hai mu Alaxan kh n nh. i vi PA khi lng t
324,5 326,9 mg v i vi IB khi lng t 171,6 173,5 mg trong mt vin nn thnh
phm Alaxan.
Gia hai mu thuc Alaxan ly t hai nh thuc khc nhau, hm lng PA v IB
chnh lch nhau khng nhiu, qua kho st hai mu ta thy rng hm lng PA v IB
trong cc mu tng i n nh suy ra quy trnh sn xut n nh.

Trang 47

Chng 4. KT LUN V XUT

4.1.

Kt lun
Kt qu ti nh lng Paracetamol v Ibuprofen trong mt s dc phm

bng phng php sc k lng hiu nng cao (HPLC) cho nhng kt lun sau:
1. Chn iu kin ph hp tch v xc nh ng thi PA v IB trong mt s
ch phm thuc dng vin nn bng my sc k lng hiu nng cao vi detector UV/Vis.
Ct tch

RP 18; 220x4,4 mm; kch thc ht 5 m

Th tch vng mu

20 l

Bc sng detector

224 nm

Nng acid phosphoric

0,01M

Thnh phn pha ng

ACN : H 3 PO 4 0,01M (97 : 3 v/v)

Tc dng

0,7 ml/pht

2. xut quy trnh chun b mu chit cht ti u.

Cn chnh xc khi lng 10 vin thuc, nghin mn thnh bt.
Cn chnh xc 0,250 g mu, cho vo bnh tam gic, thm 30 ml dung dch pha
ng, nh siu m trong 5 pht v lc trong 15 pht (v = 480 vng/pht). Lc tch phn
dung dch v phn rn. Lp li 6 ln (c th kim tra dch chit ln th 6 bng cch tim
dch chit vo h thng HPLC v kim tra peak ti thi gian lu ca cht phn tch).
Thu ton b dch chit, lc qua mng lc 0,45 m, sau cho vo bnh 250 ml v
nh mc n vch bng dung dch pha ng.
3. Xc nh LOD, LQD v khong tuyn tnh cho 2 cht PA v IB nh sau:
Tn cht

Paracetamol

Ibuprofen

LOD (ppm)

1,2

1,2

LOQ (ppm)

4,0

4,0

Phng trnh hi quy

Y = 16879x 54957

Y = 25061x + 78528

H s tng quan

R = 0,9997

R = 0,9998

Khong tuyn tnh

4,8 360 ppm

4,8 360 ppm

Trang 48

4. Kho st v phn tch 2 mu thuc Alaxan. Kt qu kho st cho thy, hm

lng PA v IB trong cc mu thuc tng i ph hp vi thnh phn ghi trn bao b.
Ngoi ra, hai mu c thnh phn tng ng nhau v n nh trong thi hn s dng.
4.2.

xut
Nh vy, vi kt qu thu c, ta thy phng php HPLC c nhy cao, lp

li tt, thch hp cho vic phn tch ng thi PA v IB trong cc mu dc phm cng
nh phn tch PA hoc IB trong cc loi thc phm n thnh phn.
Chng ti hi vng nhng nghin cu trn c th gp phn vo vic ng dng
phng php RP - HPLC ni ring v phng php HPLC ni chung xc nh cc
dc cht trong cc mu thuc mi ngy cng a dng v thnh phn v hot cht.

Trang 49

TI LIU THAM KHO

[1] Pharmacy Guide - Cm nang nh thuc thc hnh, Vietnam Edition 2008,
tr 136, 196.
[2] ng Thanh Thy (2008), nh lng ng thi Paracetamol v
Ibuprofen trong ch phm dc bng phng php o quang, NXB H Dc H Ni.
[3] ng Qunh Chi (2008), nh lng ng thi Paracetamol v Codein
Phosphat bng phng php in di mao qun, NXB H Dc H Ni.
[4] Deodhar MN et al (2009), Reverse Phase HPLC Method for
Determination of Aceclofenac and Paracetamol in Tablet Dosage Form, Research
Chem, page 37 40.
[5] Patrica C. Damiani, et al (2001), Spectrofluorometric determination of
ibuprofen in pharmaceutical formulations, Journal of Pharmaceutical and Biomedical
Analysis, Vol 25, Issue 3 4, pages 679 683.
[6] S.A Kulichenko and S.O Fessenko (2003), Determination of ibuprofen
and novocaine hydrochloride with the use of watermicellar solutions of surfactants,
Analytica Chimica Acta, Vol 481, Issue 1, pages 149 153.
[7] Nguyn Hu M v cng s (2007), Nghin cu ng dng phng php
HPLC nh lng ibuprofen trong huyt tng ch, Bo co y hc, Hc vin Qun Y,
NXB i hc Dc H Ni.
[8] Phm Thanh Huyn (2008), Nghin cu xy dng phng php nh
lng ibuprofen trong huyt tng bng sc k lng hiu nng cao, Kha lun tt
nghip, Hc vin Qun Y, NXB i hc Dc H Ni.
[9] Phm Thu Qu (2010), nh lng ng thi Paracetamol v Ibuprofen
trong vin nn bng phng php quang ph o hm, NXB H Dc H Ni.
[10] M.R. Khoshayand et al (2008), Simultaneous spectrophotometric
determination of paracetamol, ibuprofen and caffeine in pharmaceuticals by
chemometric methods, Iran, Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy, Vol 70, Issue 3, pages 491 499.
[11] Thi Duy Thn v cng s (2003), Nghin cu ng dng phng php
sc k lng hiu nng cao (HPLC) v o quang ph UV vis nh tnh v nh lng
cc hot cht trong thuc c t 2 n 5 thnh phn, Vin kim nghim thuc B Y t,
Trng i hc Dc H Ni.
Trang 50

[12] Prasanna Reddy Battu and MS Reddy (2009), RP-HPLC Method for
Simultaneous Estimation of Paracetamol and Ibuprofen in Tablets, Asian J. Research
Chem, page 70 72.
[13] Dipankar Basu et al (1998), Application of least squares method in
matrix form: simultaneous determination of ibuprofen and paracetamol in tablets,
Original Research Article Journal of Pharmaceutical and Biomedical Analysis, Vol 16,
Issue 5, pages 809 - 812.

Trang 51

PH LC
PH LC 1. Mt s sc k ca dy cht chun Paracetamol

Hnh 1.1 Sc k ca cht chun Paracetamol, C = 360 ppm

Trang 52

Hnh 1.2 Sc k ca cht chun Paracetamol, C = 240 ppm

Hnh 1.3 Sc k ca cht chun Paracetamol, C = 115,2 ppm

Trang 53

Hnh 1.4 Sc k ca cht chun Paracetamol, C = 57,6 ppm

Trang 54

Hnh 1.5 Sc k ca cht chun Paracetamol, C = 28,8 ppm

Hnh 1.1 Sc k ca cht chun Paracetamol, C = 9,6 ppm

Trang 55

Hnh 1.7 Sc k ca cht chun Paracetamol, C = 4,8 ppm

PH LC 2: Mt s sc k ca dy ng chun Ibuprofen

Trang 56

Hnh 2.1 Sc k ca cht chun Ibuprofen, C = 360 ppm

Hnh 2.2 Sc k ca cht chun Ibuprofen, C = 240 ppm

Trang 57

Hnh 2.3 Sc k ca cht chun Ibuprofen, C = 115,2 ppm

Trang 58

Hnh 2.4 Sc k ca cht chun Ibuprofen, C = 57,6 ppm

Hnh 2.5 Sc k ca cht chun Ibuprofen, C = 28,8 ppm

Trang 59

Hnh 2.6 Sc k ca cht chun Ibuprofen, C = 9,6 ppm

Hnh 2.7 Sc k ca cht chun Ibuprofen, C = 4,8 ppm.

Trang 60

Trang 61

PH LC 3. Sc k ca mu M 01

Hnh 3.1 Sc k mu M 01 tim ln 4.

Trang 62

Hnh 3.2 Sc k mu M 01 tim ln 3.

PH LC 4. Sc k mu M 11 .

Hnh 4.1 Sc k mu M 11 tim ln 2.

Trang 63

Hnh 4.1 Sc k mu M 11 tim ln 3.

Trang 64