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7/31/2019 Luan Van Thac Sy_Tuan K19

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Nin kho: 2010 - 2012

NGHIN CU XY DNG QUY TRNH

NH LNG DIACETYL TRONG CCLOI BIA BNG PHNG PHP SC

K KH GHP KHI PH

Chuyn ngnh: HA HC PHN TCH

Ngi hng dn khoa hc

TS. NG VN KHNH

Hc vin thc hin

H THANH TUN

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Bia l loi nc ung c bit c cn thp, hng

thm, bt mn v c rt nhiu ngi a chung. Trong qutrnh ln mem bia, ngoi cc sn phm ch yu l cn vCO2 cn to rt nhiu sn phm ph khc nh ru bc cao,este, andehit, glyxerin... trong c diacetyl.

Diacetyl l cht c gy km hm s trao i cht, c nhhng n s sinh sn, lng, c lm cho men to ra

mi v l, kh chu, gy au u v nh hng n h thnkinh ca ngi tiu dng.

Phng php GC/MS kt hp k thut chit pha rn(SPE) v to dn xut vi 1,2-diaminobenzen c gii hn phthin thp c nng ppb v c chnh xc cao. Do ,chng ti chn ti Nghin cu xy dng quy trnh nhlng diacetyl trong cc loi bia bng phng php sc k kh

ghp khi ph

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CHNG I. TNG QUAN L THUYT

CHNG III. KT QU V THO LUN

CHNG II. NI DUNG VPHNG PHP NGHIN CU

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1.1. TNG QUAN V DIACETYL

Gii thiu tnh cht ha l ca diacetyl v c tnh ca n.S hnh thnh diacetyl trong qu trnh ln men bia

1.2. PHNG PHP SC K KH GHP KHI PH

Trnh by nguyn l v GC/MS, mt s b phn quan trngtrong my GC/MS. Gii thiu v khi ph, c ch phn mnh

1.3. K THUT CHIT PHA RN (SPE)

Tng quan v phng php chit pha rn SPE, c ch hpph v k thut tin hnh

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2.1. NI DUNG NGHIN CU

(1). La chn cc iu kin tin hnh sc k khi ph: cttch, tc kh mang, nhit interface, chng trnh nhit

l, kiu bm mu.(2). nh gi tin cy ca phng php phn tch qua cci lng nh lp li, ng, thu hi, gii hn pht hinv gii hn nh lng.

Kho st cht lng qui trnh phn tch bng cch so snhphng php ang nghin cu vi phng php chun theoTCVN 6058:1995

(3). p dng thc t: p dng phng php la chn nh lng cc loi bia hin ang lu hnh trn th trng Tha

Thin Hu.

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2.2. PHNG PHP NGHIN CU2.2.1. Ly mu v bo qun muLy mu ngu nhin quy, nh hng, cc i l mu

c ng trong chai hoc lon kn c ngy sn xut v c

bo qun ni thong mt2.2.2. Quy trnh x l muLy khong 10-50 mL mu th cho vo cc c m c dung

tch 125 mL, thm khong 1-5 mL dung dch 2,3-hexandion v1-2 mL dung dch 1,2-diaminobenzen, un cch thy 300Ctrong khong 15 pht, cho qua ct SPE c hot ha, sau ra gii bng dung mi diclometan. Dung dch sau khi quact, c lm lm kh bng kh N2 ti cn. Ha tan cn vidung mi etylaxetat, lc qua mng lc 0,45 L v tim vomy GC/MS.

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2.2.3. Nghin cu quy trnh phn tch2.2.4. nh gi tin cy ca phng php phn tch

* Xy dng ng chun* lp li

* Gii hn pht hin (LOD) v gii hn nh lng (LOQ)2.2.5. nh gi phng php nghin cu

* Dng chun F (chun Fisher) so snh lp li ca haiphng php: phng php nghin cu v phng php

chun theo TCVN 6058:1995,* Dng chun t (chun Student) so snh hai gi tr trungbnh ca phng php GC/MS vi phng php i chiu:phng php chun theo TCVN 6058:1995

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3.1. KHO ST IU KIN SC K

3.1.1. La chn ct tchBng 3.2.So snh kh nng tch ca 2,3-dimetylquinoxalin v 2-

metyl-3-n-propylquinoxalin trn hai ct DB-5MS v InertCap

5MS/Sil

STTLoi ct

Thi gian luca pic 2,3-

dimetylquinoxal

in (pht)

Thi gian luca pic 2-metyl-

3-n-

propylquinoxali

n (pht)

H stch

1 InertCap 5

MS/Sil

7,098 8,890 1,252

2 DB-5MS 9,013 11,117 1,233

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Hnh 3.1.Sc phn tch hn hp trn ct InertCap 5MS/Sil

Hnh 3.2.Sc phn tch hn hp trn ct DB-5MS

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3.1.2. Kho st tc dng kh mang

Sau khi la chn ct DB-5MS tin hnh phn tch v ginguyn KTN nh hnh 3.2. Chng ti tin hnh tim hn hp

chun c to dn xut 2,3-dimetylquinoxalin v 2-metyl-3-n-propylquinoxalin vo my sc k kh ghp khi ph vi tc kh mang ln lt l 1,2; 1,5; 1,7 mL/pht. Kt qu phn tchc trnh by bng 3.3

Bng 3.3.So snh kh nng tch ca 2,3-dimetylquinoxalin v 2-metyl-3-n-propylquinoxalin ba tc dng khc nhau

STT

Tc dng

(mL/pht)

Thi gian lu capic 2,3-

dimetylquinoxalin(pht)

Thi gian lu ca pic2-metyl-3-n-

propylquinoxalin(pht)

H s

tch

1 1,7 8,608 9,052 1,052

2 1,5 9,047 11,097 1,226

3 1,2 10,605 12,749 1,202

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Hnh 3.3.Sc khost tc dng 1,7ml/pht

Hnh 3.4.Sc kho

st tc dng 1,5ml/pht

Hnh 3.5.Sc khost tc dng 1,2ml/pht

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3.1.3. La chn cc iu kin tin hnh sc k phn tch dn xut cadiaxetyl trong bia

Bng 3.4. Chng trnh nhit phn tch hn hp 1,2-dimethylquinoxaline v2-methyl-3-n-propylquinoxaline trn h thng GC/MS

Chngtrnh

Nhit MS

interface

(0C)

Nhit bung

tim mu(0C)

Chng trnh nhit lTc

tng nhit

(0C/pht)

Nhit t ti

Thi giangi

(pht)

Tng thigian

(pht)

1 250 250

60 1

29,330 250 0

20 300 5

2 250 250

60 1

25,020 200 1

30 300 5

3 250 250

40 1

28,510 200 0

20 250 5

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Bng 3.5.iu kin sc k kh ghp khi ph xc nh diacetyl trong bia

Thng s iu kin

Ct tch

DB-5 MS: di 30m, ng knh 0,25mm, pha tnh

cha (5%-phenyl)-metyl polysiloxan vi b dy0,25 m.

Kh mang Heli tinh khit 99,99999%

Tc dng 1,5 mL/pht

Kiu bm mu Splitless, mu c bm t ng vi th tch 1lNhit MS interface 2500C

Nhit injector 2500C

in th detect 1,2 kV

Ch ion ha va p int EI vi nng lng 70 eV

Ch Full Scan, di qut 50 500 amu

Chng trnh nhit Nhit u 400C (gi ng nhit trong1 pht);tng 100C pht n 2000C; tng 200C pht n250 0C (gi ng nhit trong5pht)

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Hnh 3.8.(a) cc mnh ph ca hp cht 2,3-dimetylquinoxalin(b)cc mnh phca hp cht 2-metyl-3-n-propylquinoxalin

Hnh 3.7.Ph Scan1,2-dimetylquinoxalinv 2-methyl-

3nPropylquinoxaline

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3.2. KHO ST QUY TRNH CHIT TCH DIACETYL VTO DN XUT S DNG CT CHIT PHA RN (SPE)

3.2.1. Qu trnh tch chit

- Hot ha ct chit: Ct chit C18 c dung tch 3 mL, cha200mg pha rn C18 c hot ha vi 3 mL metanol/nc vi tl 9/1 (V/v) tip sau l 3 mL nc ct.

- Np mu vo ct: Mu c np vo ct vi tc c

iu chnh bng p sut ca bm ht chn khng m bo tc dng khong 1mL/pht.- Ra gii :

3.2.2. Kho st th tch dung mi ragii hm lng diacetyl qua

ct C18Vilng diacetyl ban u l 200 g/L, sau khi tin hnh todnxut v cho qua ct C18 c th tch l 3 mL tin hnh khost th tch ragii khc nhau. Ktqukho st cthhin

bng 3.6.

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Bng 3.6.Kt qu kho st th tch dung midiclometan ra gii qua ct C18

Th tch dung mi ra gii (mL) 2 3 5 7

Lng diacetyl thu hi(g/L )(n = 3)

Ln 1 151,5 181.2 190.5 184.4

Ln 2 162.2 195.6 191.4 180.2Ln 3 148,4 182.5 189.5 200.5

Trung bnh 154,0 186,4 190,5 188,4

thu hi (%) 77,0 93,3 95,3 94,2

Hnh 3.9.Hiu sut thu hi diacetyl cc th tch dung mi ra gii

khc nhau sau khi qua ct C18 16


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3.3. THM NH PHNG PHP3.3.1. nnhcahthngsc kKtqukho st c trnh by bng 3.7

XX ((STT

Nng

nghin

cu

T l dintch pic

SD) STT

Nng

nghin

cu

T ldin tchpic

SD)

1

20 g/L

0,37

0,37 0,01RSD% = 1,71

13

100

g/L

0,69

0,69 0,01RSD% = 1,09

2 0,37 14 0,70

3 0,38 15 0,69

4 0,37 16 0,69

5 0,36 17 0,70

6 0,37 18 0,68

7

50 g/L

0,53

0,53 0,01RSD% = 0,97

19

200

g/L

1,07

1,06 0,01RSD% = 1,39

8 0,54 20 1,08

9 0,53 21 1,05

10 0,54 22 1,06

11 0,53 23 1,07

12 0,53 24 1,04

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3.3.2. Gii hn pht hin v gii hn nh lngThm dung dch diacetyl chun 16,5 g/L; 32,0 g/L; 64,0 g/L vo mu trng.

X l mu v tin hnh chy sc k. Tnh trung bnh ca t s chiu cao ng vipic ca hp cht 2,3-dimetylquinoxalin so vi chiu cao nhiu ng nn ca 3 lnlm song song ti mi nng kho st. Ti nng c t s S/N khong bng 10vi RSD khong 20%, ng nm trong khong 80- 110% l LOQ ca phng

php v t s S/N bng 3 l LOD ca phng php.Tin hnh th nghim lp li 3 ln nng 16,5 g/L, t kt qu thu c

hnh 3.10 v ph lc 2. Chng ti xc nh c LOD v LOQ ca phng php l: LOQ = 16,5 g/L v LOD = 5,0 g/L

PH LC 2. Bng s liuphn tch gi tr S/N tinng 5 ppb

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3.3.3. Khong nng tuyn tnhKt qu c trnh by bng 3.8. v hnh 3.11

Bng 3.8.Kt qu kho st khong tuyn tnh ca diacetyltrong bia

STTNng diacetyl

(g/L)

T l din tch pic(2,3-dimetylquinoxalin/2-metyl-3-n-

propylquinoxalin)

Phng trnh hi quy

1 20 0,37 y = 0,0039x +0,3073

R = 0,9995

y : t l din tch pic

x : nng diacetyl

2 50 0,53

3 100 0,69

4 200 1,07

5 500 2,25

y = 0.0039x + 0.3073

R2

= 0.9994

0

0.5

1

1.5

2

2.5

0 100 200 300 400 500 600

Hnh 3.11.ng biu din mitng quan gia t s din tch2,3-dimetylquinoxalin/2-metyl-3-n-

propylquinoxalin v nng diacetyltrong bia

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3.4. XY DNG QUI TRNH PHN TCHT cc kt qu kho st nghin cu, chng ti xy dng quy trnh phn

tch nh lng diacetyl trong bia bng phng php GC/MS nh hnh 3.12

20 mL mu bia 1 mL dung dch 2,3-hexandion 1ppm

To dn xut vi 1,2-diaminobenzen

SPE

Cn

1 mL dung dch

GC/MS

- Ct tch: DB-5MS- Tc dng: 1,5 mL/pht

-Nhit inject: 250o

C-Nhit interface: 250oC- in th detect: 1,2 kV-Nhit u 40oC (gi ng nhittrong 1 pht); tng 10oC/pht n200oC; tng 20oC/pht n 250oC(gi ng nhit trong 5 pht)- Di qut: 50500 amu

- Cch thy 300C trong 15

- Ra gii bng 5 mL CH2Cl2- Thi N2ti cn

- nh mc 2 mL Etyl axetat

Hnh 3.12.S quytrnh phn tch xc nhdiacetyl trong bia bng

phng php GC/MS

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3 5 P DNG THC T 21


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3.5. P DNG THC T3.5.1. Kim sot cht lng qui trnh3.5.1.1. lp li v thu hi

Chunb cc dung dchchun diacetyl cho vo mutrngvi 3 nng380 g/L; 470 g/L; 550 g/L v dung dchnichun 2,3-hexandion nng 1000 g/L. Minng lm song song 3 mu. Tin hnh phn tch theoqui trnh xy dng. Ktquuc trnh by bng 3.9.

Bng 3.9.Kt qu kho st lp li v thu hi ca quy trnh phn tch

Lng thm

vo (g/L)

Lng tm thy (g/L)/Rev%RSD%

(n = 3)Rev%

380,0 325,2/85,58 321,5/84,61 327,6/86,21 0,95 85,47

470,0 431,2/91,74 421,5/ 89,68 439,2/93,45 2,06 91,62

550,0 528,2/96,04 521,2/94,76 524,5/95,36 0,67 95,39

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3.5.1.2.So snh phng php nghin cu vi phng php hin hnh theoTCVN 6058-1995 (UV-Vis)

Tin hnh phn tch hm lng diacetyl trong mu bia Hue beer Export(chi tit v mu nu bng 3.11) bng hai phng php: phng php sc

k kh ghp khi ph GC/MS v phng php quang ph hp th phn tUV-Vis. Kt qu phn tch c trnh by bng 3.101X2X

Bng 3.10.Kt qu phn tch diacetyl trong bia bng phng php GC/MS vUV-Vis trong mu bia B30

MuGC/MS UV-Vis

Hm lngdiacetyl (ppb)

S liu thngk

Hm lngdiacetyl (ppb)

S liu thngk

1 48,5

= 48,5

S1 = 0,32

(n = 5)

47,7

= 49,0

S2 = 1,04

(n = 5)

2 48,2 49,9

3 48,7 50,2

4 48,3 48,6

5 49,0 48,5

1X 2X

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2 2

2

2 2

1

1, 04

0,32

S

S

1 2

2 2

1 2

1 2

X X

S Sn n

22 2

1 2

1 2

2 22 2

1 2

1 2

1 2

2 5

1 1

S S

n nf

S S

n n

n n

*Nhn xt:- Ta c Ftnh =

Nn Hai phng php c lp li khc nhau vi p < 0,05. Hayni cch khc, phng php GC/MS c lp li tt hnphngphp UV-Vis vi p < 0,05.

- Xt ttnh = = 1,03 v khi bct do fc tnh li

Do ttnh = 1,03 < t (p = 0,05; f= 5) = 2,57.

Nn ktqu trung bnh ca hai phngphp tngng nhau(p < 0,05).

= 10,56 > F (p = 0,05; f1= f2=4)= 6,39

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3 5 2 p dng thc t 24


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3.5.2. p dng thc tp dng qui trnh c xy dng v thm nh c tin hnh nh

lng mt s mu bia ang lu hnh trn th trng thnh ph Hu (Chi titv mu nu bng 3.11) thu c kt qu bng 3.12.

Bng 3.11. Thng tin chi tit v cc mu bia

ST

T

K hiumu

Tn muNng

cnNgy sn xut

1 B25 Tiger beer 5% 01/08/2012

2 B26 Huda beer extra 4,9% 06/02/2012

3 B27 Biere Larue export 4,5% 01/08/2012/SV

4 B28 Festival beer 4,5% 07/03/2012

5 B29 Corona extra (Mexico) 4,6% 05/03/2012

6 B30 Hue beer export 5% 10/12/2012

7 B31 Tiger crystal 4,6% 26/07/2012/AE

8 B32 Huda Beer 4,7% 19/02/2012

9 B33 Heineken 5% 04/08/2012/HE

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Bng 3.12.Kt qu phn tch mt s mu bia thc t bng GC/MSSTT

K hiu muHm lng

diacetyl (ppb)

So snh vi mc qui nhtheo QCVN 6-3: 2010/BYT

1 B25 47,8 t

2 B26 47,2 t

3 B27 58,0 t

4 B28 78,5 t

5 B29 46,2 t6 B30 48,5 t

7 B31 39,1 t

8 B32 68,5 t

9 B33 49,0 t

Kt qu bng 3.12 cho thy: pht hin c diacetyltrong cc loi bia ang lu hnh trn th trng Tha Thin Hu.Song hm lng ca chng u t yu cu so vi quy nh hin

hnh ca B Y T (


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Qua qu trnh nghin cu v kho st. Chng ti t c mt s kt

qu sau:1. kho st v xy dng qui trnh phn tch diacetyl trong bia bngphng php sc k kh ghp khi ph (GC/MS). Trong :

- X l mu bng phng php chit pha rn SPE s dng ct C18 vith tch ra gii l 5 mL diclometan.

- iu kin chy sc k c th nh sau: Ct mao qun DB-5MS: chiudi 30 m, ng knh trong 0,25 mm, pha tnh cha (5%-phenyl)-metylpolysiloxan vi b dy 0,25 m; kh mang Heli tinh khit 99,99999%, tc dng 1,5 mL/pht, k thut bm: Splitless vi th tch tim mu 1l,nhit MS interface: 2500C, nhit injector: 2500C. Chng trnhnhit : nhit u 400C (gi ng nhit trong 1 pht); tng 100Cpht n 2000C; tng 200C pht n 250 0C (gi ng nhit trong 5pht). Ch ion ha va p in t: EI vi nng lng 70 eV. Ch Full Scan, di qut: 50 500 amu.

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- Vi cc iu kin th nghim trn, qui trnh t c

n nh tt, lp li cao vi RSD < 2,06% (n = 3), ng tt vi thu hi Rev t 85,5% - 95,4%. Phng phpt c gii hn pht hin thp (5 ppb) v khong tuyntnh rng (20 500 ppb) vi R2 = 0,9994. Kt qu phn tch

c so snh vi phng php hin hnh theo TCVN6058:1995 cho kt qu ph hp.

2. p dng thnh cng quy trnh xy dng c phn tch hm lng diacetyl trong 09 mu bia ang lu

hnh trn th trng Tha Thin Hu. Cc kt qu cho thy,hm lng diacetyl trong cc loi bia trn u t yu cu sovi quy nh hin hnh ca B Y T

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THEO DI

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