TRNG I HC BCH KHOA TP.

HCM
KHOA K THUT HA HC

TIU LUN

GII HN PHT HIN

GVHD: TS. HUNH KHNH DUY

HVTH: PHM S NGUYN
MSHV: 13050192

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 MC LC

CHNG 1: KHI NIM V THM NH PHNG PHP ................................................ 3

1.1. Khi nim v thm nh phng php.................................................................................... 3
1.2.Thm nh phng php tiu chun ........................................................................................... 3
1.3.Thm nh phng php khng tiu chun................................................................................. 4
1.4. Thm nh li............................................................................................................................. 5
CHNG 2: THM NH PHNG PHP PHN TCH HA HC .................................. 7
2.1. Thm nh phng php ha hc ............................................................................................ 7
2.2. Gii hn pht hin ..................................................................................................................... 7
2.3. Cch xc nh gii hn pht hin ............................................................................................ 8
2.4.Gii hn nh lng ................................................................................................................... 11
2.5.Cch xc nh gii hn nh lng ............................................................................................. 12
2.6.Tnh c hiu/Tnh chn lc....................................................................................................... 14
TI LIU THAM KHO ............................................................................................................. 16

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 CHNG 1: KHI NIM V THM NH PHNG
PHP
1.1. Khi nim v thm nh phng php

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. (fitness for the purpose). Kt qu ca thm nh phng php c th c
s dng nh gi cht lng, tin caayj 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.

Hin nay nhiu thut ng khc nhau c s dng ch khi nim trn, nh
nh tr phhuwong php, nh gi phng php, xc nhn gi tr s dng ca
phng php, ph duyt phng php. Tt c cc thut ng ny u l cch gi
khc nhau ca thm nh phng php (method validation)

Phng th nghim thng s dng nhiu phng php khc nhau. 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 (non-

standard/alternative/in-house methond): l cc phng php do phng th
nghim xy dng, phng php theo hng dn 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 (non-standard
method)

Phng php do phng th nghim t xy dng mi trc khi a vo s dng

quy trnh.

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.

1.2.Thm nh phng php tiu chun

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 Trc khi p dng mt phng php phn tch cn c cc chng minh rng
phng php p ng yu cu t ra, tc l phng php gii c thm
nh. Yu cu ny khng ch cho cc phng php th ni b m cn cn cho
cc phng php tiu chun. Vic thm nh phng php tiu chun v phng
php ni b c s khc nhau, do cn ch khi lp k hoch thm nh. C
hai yu cu ch yu ca vic thm nh phng php tiu chun:

Phi c kt qu thm nh ca phng php tiu chun, v kt qu ny phi ph

hp vi yu cu ca phng th nghim.

Phng th nghim cn m bo c th t c cc thng s c m t trong

phng php tiu chun.

Theo yu cu ca ISO 17025, khi cc phng th nghim p dng cc phng

php tiu chun cn c h s nh gi cc iu kin c bn, cc ngun lc theo
yu cu ca phng php th v vic t c kt qu th nghim c chnh
xc nh phng php yu cu hoc nh mong mun ca phng th nghim. i
vi cc phng php th ban hnh m khng c d liu v chnh xc th
phng th nghim phi xc nh d liu chnh xc ca php th da trn d
liu nghin cu th nghim

xy dng cc bc thm nh phng php tiu chun cn kim tra:

1. Phng php c thm nh hay cha, thm nh ton b hay mt phn

2. Nn mu c ging nhau hay khng
3. Khong lm vic ca phng php c ph hp vi yu cu ca phng th
nghim hay khng
4. C cng loi thit b ( hang sn xut, model) hay khng? Phng php tiu
chun c cho s dng cc loi thit b khc khng ?
5. C nhng lu g c bit ca phng php tiu chun m phng th
nghim khng th p ng khng ?

Nu mt trong cc yu t trn khng ph hp, th phng th nghim cn thc

hin cc php th nh gi li phng php. Cc kt qu nh gi ny cn
phi tng ng vi cc kt qu thm nh ca phng php chun, nu khng
cn phi thm nh li ton b phng php.

1.3.Thm nh phng php khng tiu chun

i vi phng php khng tiu chun, vic thm nh phi tri qua nhiu bc
hn, bt u t qu trnh nghin cu kho st phng php, ti u ha phng
php n khi hon thin phng php. Thm nh phng php l mt yu cu

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 bt buc phi thc hin i km vi vic php trin phng php mi v p dng
cc phng php khng tiu chun vo thc hin thnh thng quy. Cc bc
tin hnh thm nh bao gm:

1. Xy dng SOP d kin ( thoe cc ti liu tham kho hoc theo cc nghin
cu xy dng phng php mi)
2. Xy dng cng thm nh bao gm:
a. Xc nh thi gian v ngi thc hin
b. Cht cn phn tch: tn cht, d on hm lng trong mu
c. Xc nh i tng thm nh: nn mu
d. Xc nh mc ch cn phi t: yu cu v gii hn cho php, cn t
LOD, LOQ, chnh xc
e. Xc nh cc thng s cn thm nh v khong chp nhn
f. Xc nh cc th nghim cn thc hin
3. Kim tra cc iu kin cn cho cng vic n nh
a. Cc yu cu v trang b
b. Ha cht, thuc th
c. Mu th nghim
4. Thc hin thm nh
a. Cc php th thm nh s b
b. Thay i cc thng s ca phng php
c. Thc hin thm nh ton din
5. Hon thin SOP ca phng php
6. Bo co thm nh: cn c cc thng tin sau
a. Tn ngi thm nh, thi gian thm nh
b. Tm tt phng php: nguyn l, thit b, ha cht, quy trnh
c. Cc kt qu thm nh
d. Cc yu cu p ng a phng php vo thc hin thng
xuyn: kim tra tnh tng thch ca h thng mu, mu QC, c
lng khng m bo o ca kt qu
e. Xc nh cc thng s v thi gian cn thm nh li

1.4. Thm nh li

Cng vic thm nh khng ch thc hin mt ln khi pht trin phng php ban
u m cn thc hin trong sut qu trnh p dng. V a s cc iu kin thc hin
phng php c s thay i trong sut qu trnh p dng. V d nh c s thay i

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hoc m rng i tng p dng, thay i a im phng th nghim, thay i
nhn vin, thay i thit b( p dng trn cc thit b cng loi khc) thay i cc
iu kin v tin nghi mi trng, thay i v dung mi ha cht thuc th, nhng
thay i nh khc( v d nhit ct phn tch, pH pha ng) Trong trng hp kt
qu phn tch mu kim tra QC hoc kt qu nh gi s ph hp ca h thng nm
ngoi gii hn cho php th phng php cng cn phi thm nh li. Phng th
nghim nn phi hp qu trnh tnh n inh vi qu trnh thm nh li cc
phng php phn tch hng ngy

Cc thng s cn thm nh li ph thuc vo mc nh hng ca cc thay i

n cc thng s phng php. Thng thng tin hnh thm nh cc thng s c
bn nht nh trong trng hp thm nh cc phng php tiu chun. Tuy nhin
nu nhng kt qu thm nh ny c s sai khc nhiu so vi kt qu thm nh ban
u th cng cn thc hin thm nh li ton b

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 CHNG 2: THM NH PHNG PHP PHN TCH
HA HC

2.1. Thm nh phng php ha hc

Thm nh phng php l mt cng vic rt kh khan, nhm chn v tn km

tuy nhin li l mt ni dung quan trng nh hng n chnh xc v kt qu
phn tch. Cn cn nhc mc ch yu cu ca tng phng php v ngun lc
la chn thng s thm nh cho ph hp.

Theo cc quy nh ca USFDA, AOAC, USP v ICH, i vi cc phng phpas

phn tch ha hc cc thng s cn thm nh bao gm:

Tnh c hiu, tnh chn lc

Khong tuyn tnh v ng chun
Gii hn pht hin
Gii hn nh lng
ng
chm
vng

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.

2.2. Gii hn pht hin

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
L mt i lng c lin quan n tnh n nh ca php o. LOD ph thuc vo:
Cp hin i, iu kin vn hnh thit b(dao ng nhit , p sut, in p)
Phng php phn tch ( x l mu, mt mt, nhim bn mu, nhiu nn)
Tay ngh ca phn tch vin
Trong thc t thng gp : LOD ca thit b ( gii hn pht hin ca thit b: IDL:
instrumental detection limit) v LOD ca phng php ( Gii hn pht hin ca
phng php-MDL: method detection limit)

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IDL: l gi tr nng ca cu t cn phn tch c th to tn hiu 5 ln tn hiu
nhiu ca thit b hoc 3 ln lch chun b vn hnh c n nh hay khng, n
bao gm cc loi nhiu t linh kin c- in t ca thit b, iu kin vn hnh my
v iu kin mi trng xung quanh v thng c c lng qua cc dung dch
chun. IDL c xc nh bng thc nghim
Gii hn pht hin ca phng php MDL: method detection limit : l gi tr nng
ca thnh phn cn phn tch c tin hnh phn tch theo mt phng php,
to ra mt tn hiu vi xc sut 99% khc bit vi mu trng
Quan h gia IDL:MDL xp x 1:4

2.3. Cch xc nh gii hn pht hin

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 tram s ln pht hin ( dng tnh) hoc
khng pht hin (m tnh)
V d

Trong v d trn, vi nng di 100 ppm kt lun dng tnh khng cn chc
chn 100%, gii hn pht hin trong trng hp ny l 100 ppm.
3.2 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:

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Trong

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 x, v lch chun SD

nh gi LOD tnh c : tnh R= x/ 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 dch th
dng v lm li th nghim v tnh li R

Da trn t l tn hiu 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

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Nhiu ng nn c tnh v hai pha ca ng nn v tt nht l tnh nhiu
ln cn hai bn ca pic, b rng mi bn ti thiu gp 10 ln chiu rng ca pic
ti na chiu cao

LOD c chp nhn ti nng m ti tn hiu ln gp 2-3 ln nhiu ng

nn, thng thng ly S/N =3

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=(3,3xSD)/a

Trong : 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 v tnh SD

Da trn h s chn ca ng chun, lm nhiu ln tnh SD ca gi tr b

Da trn lch chun ca khong cch cc gi tr o thc vi ng chun.

Xc nh theo quy tc 3

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 Theo di LOD hng ngy

LOD bin thin vi chc %: chp nhn c

LOD bin thin vi tram %: cn xem xt tng th thit b, iu kin lm vic, tay
ngh nhn vin

2.4.Gii hn nh lng

LOQ 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

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 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.

Mi quan h gia LOD, LOQ v khong tuyn tnh c m t bng hnh
di y:

2.5.Cch xc nh gii hn nh lng

Vic b tr th nghim xc nh LOQ thng kt hp vi tnh LOD. C nhiu

cch tnh 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

V d: xc nh gii hn pht hin v gii hn nh lng ca phng php

phn tch methanol trong ru, thc hin phn tch mu trng ( mu c hm
lng methanol rt thp, gn vi gii hn di ca ng chun) thc hin phn
tch 10 ln lp li v tnh gi tr trung bnh v lch chun, thu c cc kt
qu su:

Phng trnh ng chun: y= 13,227 x + 0,004

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Nh vy: LOD = 13,61 + 3x1,75 =18,86 mg/l

LOQ = 13,61 + 10x1,75=31.11mg/l

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 ly S/N = 10

V d: Xc nh LOD ca phng php phn tch axit bezoic trong nc gii

kht bng HPLc, ngi ta thc hin phn tch cc mu trng c thm chun axit
benzoic cc nng thp di gii hn thp nht ca ng chun. Tnh chiu
cao ca pic sc k ( H l tn hiu S) v chiu cao ca nhiu ng nn ( h= 2N)

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 v hai pha ca pic. Thu c cc kt qu nh sau: Da trn ng chun

Cch tnh tng t nh trong phn LOD nhng theo cng thc sau:

LOQ= (10xSD)/a

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 . Xem thm phn chm v yu cu RSD % cho tng nng d c th

2.6.Tnh c hiu/Tnh chn lc

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 C th trong
php phn tch nh tnh l phi chng minh c kt qu l dng tnh khi
c mt cht phn tch, m tnh khi khng c mt n, ng thi kt qu phi l
m tn khi c mt cc cht khc c cu trc gn ging cht phn tch. 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 qun n vic xc nh ch mt cht phn tch

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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

xc nh tnh c hiu/chn lc ca phng php nh tnh, nh lng cn

b tr cc th nghim 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.

Phn tch mu th hoc mu trng thm chun hm lng cn 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, cch ny thng p dng
i vi phng php sc k. Sau khi chun b mu 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 c hiu/chn lc.

Phn tch mu khng c cht phn tch nhng c cht cu trc tng t cht
phn tch. 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)

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 TI LIU THAM KHO

[1] Thm nh phng php trong phn tch ha hc v vi sinh vt

[2] Hng dn x l v phn tch s liu (Vin khoa hc v cng ngh mi trng)

[3] Gii hn pht hin ( Limit of detection)

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