Tran Qudc Toan vii Dig T^p chi KHOA HOC & CONG NGHE I06{06)-:
XAC BINH DONG THOI VITAMIN C, XITRAT VA OXALAT
TRONG N l / 6 C TIEU BANG PHUONG PHAP DONG HOC TRAC QUANG Tran Quoc To^n , Mai \ u 3 n Truong Tnr&ng DQI II()C .Sirpham - Dll Tlidi Nguyen TOM T A T
Bai bao nay thong bdo k^I qua nghien ciru xac dnih dong thdi vitamin C. xilrat va oxalai trong nirac tieu bang phirong phap dpng hpc IrSc quang sir dung Ce(IV) lam chat oxi hoa. Cac tht nghiem do do hap thu quang ciia cac dung djch diroc li^n hanh sau 20 gia) ke Iir khi them Ce(IV) d budc song 320nin, vdi dung dich so sdnh la H2SO4 0,7M trong khoang thoi gian do 20 - 120 giay. Cac s6 lieu dp hdp thu quang diryc xir li bdng phdn m6m MATLAB d^^a tren phuong trinh hdi qui da bien da lap. Cac ket qua Ihyc nghiem cho lhay: gidi han phat hi?n vitamin C, xitrat vd oxalat ldn luot la 7,75.10''|V1, T.aS.lO'^M va 7.I7,I0"''M; gioi han djnh luong vitamin C, xitrat va oxalat ldn tu-gt la 2.5,10'''M. 2,46 ! (1""M va 2.39, i O" M
Til" khoa dong hoc irac quang, vilamm C, xitrat, oxalai. Cc(IT). mrac lieu.
MO DAU
Trong dieu tri benh sdi than, viec theo doi ham lugng \ itamin C. axit \itric va a.\it o\a!ic trong nirdc tieu cua benli nhan rat can thiet, bdi chiing la nh&ng chat chii yeu tao nen soi than va thudng cd mat dong thdi trong nirdc lieu. De xac dinh dong thai ham luang vitamin C, axit xitric \a axit oxalic ngudi ta phai tien hanh tach toai roi mdi xac dinh bang phuang phap circ pho xung vi phan, phirang phap von-ampe, phirang phap sdc ki long hieu nang cao (HPLC),.... Cong viec nay ton nhi^u thai gian, hda chat hoac ddi hoi phai sir dung cac thiet bj hien dai.
Trong bai bao nay chimg toi sir dung phiruiig phap dpng hoc trac quang ket hap vdi thuat toan hoi qui da bien dita tren phiin mem Matlab de nghien cuu xac dinh dong thdi vitamin C. .\itrat va oxalat trong mlu nuac tidu ma khong can phai tach loai chiing ra khoi nhau trudc khi tien hanh phan tich.
THU'C NGHIEM Hoa chat, thiet bj
- Cac hoa chSt sir dung dlu la toai tinh khiet phan tich (P.A.): Axit ascobic (C^HRO,,), natri oxalat (Na.C^Oa). Natn xitrat (Na.,C„H507), Ce(S0j)2.
' Tel 0978553908. Eimiii qiioctoanid71 Sdr^imnl ct
- May do quang UV-VIS 1700 PC - Shimazu Phuong phap nghien cu'u
Cac chat vitamin C, xitrat va oxalat phan irng vai Ce(IV) trong moi inrang axit se tam giam nong dp Ce(IV) vui loc do khac nhau. Neu theo doi toe do phan irng a thai diem dau bdng each do bien thien do hap thu quang ciia dung dich Ce{IV) tai X = 320 nm theo thai gian thi co the dinh lirong dirac cac ion nay bang phirong phap dong hoc trac quang sii dung tinh toan theo phuang phap tga.
Vi toe dp phan irng ciia cac ion can xac dinh vai Ce(IV) la khac nhau nen bien linen do hap thli quang ciia dung djch Ce(lV) theo thai gian se phu thuoc khac nhau vao nong dp cac ian va loai ion. Dira tren tinh chat na\' co the xac dinh dong thai 3 ion theo phirong phap hoi qui da bien tuyen tinh (sau khi da kiem tra tinh cong tinh), sir dung cac thuat toan binh phuang toi thieu nhir binh phuang toi thieu neng phan (PLS). hoi qui thanh phan chinh (PCR), su dung phan mem MATLAB, KET QUA VA TIIAO LUAN
Khao sat cac dieu kicn toi uu MI cac yeu to anh hirong den phep do quang.
De xac dinh dong thai vitamin C. xitrat \a oxalat sir dung Ce(IV) lam chat o\i hoa can xac djnh cac dieu kien toi uu cho phep do quang, Chiing toi da tien hanh khao sat va nit
Tran Quoc Toan vo Dig T9P chi KHOA HQC & CONG NGHE 106(06): 3-8 ra cac dicu kien toi uu cho phep do dpng hpc
trac quang la: Birac song img vai dp hap th\i quang c^rc dai ciia dung dieh Cc(IV)
^m.,v = 320 (nm): H^SO., 0,7M: kiioang thai gian do dp hap (liu quang ctia cac dung djch hon hgp la 20 - 120 giay; giai han phal hien Mlainm C. xitrat va oxalai liin lii'pt la 7,75.10' ' M . 7 .'.8.tO-^M Vii 7.17.I0"M giai han dinh luong vitamin C, xitrat \ii oxaial tail krirl la :.> 1 ()""M. 2,46.1 0""M va 2.39.! 0 ' ' M Tilth chai cong tinli \a su tuan Ihco djnh luat Bugtie - Lambe - Uia ciia dung djch lion hgp
\ itamin C. xitrat va oxalat trong toan bg khoang thai gian kliao sat dira vao he so tuang quan R \;i tic so goc ciia cac diruiig tioi qui tuyen tinh bieu dicn hicu dg hap thu quang AA ciia cac day dung djch Iren theo nong dp ciia 1 cau lu Ket qua khao sat cho thay do hap thu quang cua dung dich co tinh cong iinh tren toan bg khoang thoi gian do va luan theo dinh luat Buger -Lambe- Beer (he so goc ciia cac diraiig hoi qii\ (1). (2), (3), (4) la gan bang nhau, he so tirang; quan R deu lan han 0.999 trong cac tru0iig hgp).
Hinh 1; Duvngly'ii qtn iirn'ii linh hicii diC'n sirllniac aici hieu do hdp ihu quang vd nong do oxaial lilii khong CO imlxilral vd vilamm t' (lir duai len tren
str phu ihugv A A, j theo nong do oxaial) X a c d i n h diing t h o i v i t a m i n C , x i t i a l vii oxalat ti'ong hon h u p t u p h a
Xd) c/inig phuung Irinh tliiwig chudn du hien tie dung dich chudn va ihi nghiem kiC'in chirng linh phil hap cuu phuvng irinh hoi qui Cac mo hinh quy da bien tuyen tinh dira tren ma Iran nfing dp g6m 27 dtmg djch chuin
chira dong thoi 3 chat can xac dinh co nong dg nam trong khoang tuyen da khao sat.
D6 kicni ehiing tinh phii hgp ciia phugng trinh hoi qui ta chuan bj ina Iran gom 10 dung djch chira dong tiiai 3 chat can xac dinh co nong dp nam Irong khoang iuy6n tfnh da khao sat.
Sau 20 giay ke lir khi them Ce (IV) tien hanh do dg hap thu quang cac dung djch tren a birae song 320nin. vcn dung djch so sanh la HiSOi 0,7M trong thai gian 50 giay, moi giay ghi mgt gia irj do hap thu quang. Luu ket qua dirdi dang ma tran va chuyen so lieu vao phan mem''Jkaatlab tic tinh loan. Ket qua tinh loan sai s6 eiia phep xac dinh ham lugng vitamin C, xitrat va oxalat dirge trinh bay d bang I.
Nhan xet: qua ket qua thu duac a bang 1 chiing toi nhan iha\ khi xac dmh nong do vitamin C, xitrat va oxalat trong cung hon hgp bang phuo'ng phap CLS va PLS cho sai so lan a hau het cac mau. con phuang phap PCR cho sai so ntio hon rat nhieu. Nong do cac chat thu dugc khi phan tich theo phuang phap PCR dcu c6 sy sai khac khong dang ke so vai nong do cac dung dich chuan da pha (sai so tuang doi trong pham \ i < ± 20%), dac biet o.xalat cho sai so ttrang doi rat nho. Dicu nay co the giai thich la do trong phircmg phap CLS. anh hirdng ciia nen mau den do hap thu quang rat Ion do \ ICC do sir giam dg hap thu quang theo thai gian nen sir sai khac cua ma Iran he so a trong piiirong Irinh hoi qui da bien dang y= a+bx la dang kO trong khi thuat loan CLS thi phai xem a=!:0. Con doi \6\ phirang phap PLS. hai ma tran tri so va trgng so dugc tach ra tir ma Iran do hap thu quang Iheo thai gian va ma tnin nong dd 3 cau tir nh^ doi hoi phai cd Iinh cgng linh cao \ e tin hieu dp hap thu quang, Trong trirdng hgp phan iing xuc tac thi tinh cdng tinh chi tuan tliii khi tinh hieu sg do hap thu quang Ihco thoi gian de lam sd lieu ma Iran \ . Vi vay hai phirang phap CLS va PLS khdng thich hop khi ,\u ly tin hieu Iheo kieu bicn thien A theo t. Nhu vay de xac dmh ddng thoi vilamm C. xitrat va oxalat trong mau nirdc lieu liiirc te chimg tdi chgn phuang phap PCR dg phan tieh.
TrSn Ou6c To^n .•,1 Dig
Bang 1. Sai -TO cac phip xdc
STT 1 2 3 4 5 6 7 8 9 10
Sai so xic djnh vitami CLS
-104,83 -71,81 -0.35 -109.17 -189.79 -119.82 -60.26 -40.41 -87.10 -78.83
PLS 288749.10 -124279.00 -108521.00 419734.80 421758.70 376343.70 268017.60 216594.60 120824.40 129335.50
Tap chi KHOA HOC & CONG NGHE
^inh ham in C (%) PCR -8,58 10,15 12,33 -15,53 -11,60 10,77 -15,39 10,31 15,16 18,73
luo'ng vilamin C. xitrat va Sai so
CLS 42.93 -31.67 -46.43 -38.93 94.41 84,06 4.84 29.48 22.05 31.25
\&c dinli xitrat PLS -65466.20 37534.94 40980.56 -126919.00 -239147.00 -256058.00 -60764.80 -98232.40 -65787.00 -58666.70
oxaial II {%)
PCR 9,09 -9,88 -17,33 -2,54 -7,27 -9,06 -2.49 8,54 -10,72
8.43
106(06)'3-8
-ong hiin hop man eld pha Sai so xac dinh oxa
CLS 5.50 3.73 -0,67 9.15 16.05 14.94 0.00 0.12 -0.18 5-25
PLS -4693.02
8085.86 8825.71 -5454,05 -5703.41 -6108,26 -4358.68 -5285.55 -3933,93 -4204,44
ilat (%) PCR 3,95 -5,S9 -1,20 7,49 13,38 12.47 2,06 -1,17 -3,78 1,66 Trong do: CLS: Phuang phdp hinh pinrang lot
101 ihieu tirng phdn , PCR. Phuang phdp hoi qui cat Anh hirdng cuu cdc chdt cdn ira d(}n phep .xdc dinh vd klui ndng loai In'r
Trong nudc tieu thirdng co mat cac chat nhu ure. Mg''. C a ' \ CL, SO4'" , O ngudi binh Ihirdng, trong nirdc tieu 24 gid cd 100-300mg Ca''; 4.16-4.97mg Cl'; 60-120mg Mg-'; 0,72- l,32g SO4"" va 20-40g ure [1] Di khao sat anh hudng ciia cac chat can trd ta do do hap thu quang theo thdi gian eua dung dich chua ion can trd va ion xitrrat Tir do hap thu quang Iheo Ihdl gian ta tinh hieu do hap thu quang (trong 10 giii} dau phan ung ke tii them Ce(IV)) cua dung dich hdn hgp Ce(IV) - xitrat (AA). va eae dung dich hdn hgp Ce(IV) -xitrat - chk can trd (AA ), rdi dya vao sai sd tuang ddi d^ k6t tuan nguong anh hudng cua chSt can trd Idi phep do. Ket qua thue nghiem cho th3y cac chdt: Cl. SOj"*, Mg" , ure vdi mirc sai so tirong ddi < 15% (mirc chap nhan dirge) khdng anh hudng tdi phep do, rieng ion Ca"' anh hirdng tdi phep xac djnh nen can phai dirge tach toai ra khoi mau nghien eiiu.
Khao .sdi kho ndng hai Irir ion canxi ra klini mdu nghien ct'ru
Di toai trir ion canxi ra khdi mdu nghien ciiu ta dung phuong phap trao doi ion. Phuang phap nay dira tren kha nang trao ddi ion Ca"*
tren cot trao doi calionil.
Ihieu ihdng ihu&ng: PLS , Phuang phdp hinh phuang hi lir chinh
De danh gia hieu suat thu hdi ciia cgt trao ddi calionit ta tien hanh them chuan bang lugng chinh xac da biet ndng do vitamin C. xitrat va oxalat vao mau nirdc lieu sau do cho hon hgp them chuan di qua cdt trao ddi cation, dinh mirc va dem phan tich lai Kk qua cho thay hieu suit thu hoi vitamin C, xitrat va oxalat ciia cgt trao ddi cation theo phuo'ng phap PCR cao, nhu vay viec tach loai ion Ca'" trong mau nudc tieu bang cgt trao ddi cation cho thay khdng anh hudng tdi ham luang vilamm C, xitrat va oxalat trong mau nudc lieu.
Ddnh gid do chum cita phuvug phdp trong phdn lich mdu ihuc le
Chiing tdl tien hanh dinh lugng ddng Ihdi vitamin C, xitrat va oxalat trong cimg mot mau thuc te Lay mgt mau nudc tieu that (sau khi da dirge tach Ca"" bang cdt trao ddi calionit, va pha loang 10 lan bang dung dich H2SO4 dac sao cho axil cd ndng do cudi la 0.7M) cha vao 5 binh dinh mirc 25 ml, moi binh lOmI mau that, them lOinl dung djch Ce(IV) S.IQ-^M lan lugt vao mdi binh va dinh mirc tdi vach dinh mirc bang dung dich H2SO40,7M, Sau 20 giay k^ tir khi them dung dich Ce(IV), ta lien hanh do dd liap thu quang cac dung dieh tren d bude sdng 320niii, vdi dung dich so sanh la H7SO4 0.7M trang thdi gian 50 giay, moi giay ghi mdt gia tri do hap thu quang.
Tran Quoc Toan vd Dig Tap chi KHOA HOC & CONG NGHE 106(06): 3 - 8
Ci'ic sd lieu dd hap Ihu quang dirge chuyen vao phan mem M A T L A B v d i chuang Irinh da lap sh^ dc tinh niing do dira iren pinrang trinh hoi qui da bicn dii l(ip, Kcl qua thu dugc d hiing 2
Kcl qua khao siit cho lliii_\ phirong phiip tidi qui c<4u tir chinh PCR cho dg lap cao cliii vdi cii vilaniinC. xilrat \ i i tliic biel la oxalat, Sai so lirang doi RSD ciia phep xiic d|nli vitamin C (ir 10,37% den 10.63%, ciia phep .\ac djnh
\ i l r a r lir 5.39% dOn 7.69 % va ciia phep \;ic dinh oxalaltii 1.41% den 1,77%.
Ddnh gid dd dimg cua phuong phdp irong phdn IILII mdu ihuc le
Dc diinh gia do dimg ciia phirang phap PCR irong phan tich miiu nudc lieu Ihuc (c la diinh gia Ihdng qua lugng them chuan va dg Ilui lioi. bang each Iticm Itrgng chinh xac vitamin C. xitrat va oxalat vdi ndng do biet trirdc vao mau nudc lieu va lien hanh phaii u'cli tai.
Ndng do tugng them chuan va dd thu hdi linh Ihco phuang phap PCR duac Irinh bay d biing 3. Ket qua d bang 3 cho lhay phuong pliiip hdi qui cau Iir chinh PCR cd do thu hdi tir 77,5%) dt*n 79.0% ddi vdi vilamm C, tir 93.5% den 9 4 . 1 % ddi vdi .xitrat. tir 95.2% den 100.0% doi vdi oxalat. Dieu dd cho thay phuang phap PCR cd do diing Idt cho cac chat can phan Uang 2. Kc! cjuci kluio .sdi du
lich, dac biijt ta oxalat. Mgt trong nhiing nguyen nhan lam cho dg dting ciia vilamin C Ih<4p han so vdi xitrat va oxaial la do ndng dp vitamin C kem dn dinh, vitamin C de bj phan luiy bdi anh sang, nhiel dd, dung djeh vitamin C rat dc bj oxi hoa bdi oxi khdng khi.
\ : i c djnh done thdi vitamin C , xitrat va oxalat lroti[> cac m a u thirc tc
Mau nudc lieu dugc lay vao chai 0,5 lit sau dd dem Igc bang giay lgc va tach loai ion Ca""* •"
Iren cdi calionit. Dung dich mau nude lieu sau khi di qua cdt calionit dugc dinh mire trong binh lOOmt de phan tich. Ket qua phan lich thu dugc d bang 4.
Ket qua thu dirge eho thay ham lugng vitamin C, xitrat va oxalat trong cac mau nudc lieu thuc te Ircn deu nam Irong pham \ i eho phep [ I ] V i chira cd phirang phap phan tich tieu chuan ddng Ihdi ca ba ehi tieu nay de so sanh nen chua cd ket luan so sanh phirong phap nghien eiru vdi phuang phap tieu chuan.
Nhirng kcl qua thuc nghiem danh gia hieu suat thu hdi \ i i do lap tai, dd dimg cho thay co the ap dung phtrong phap nghien ciru de xac dinh nhanh va tin ca\ ham lirgng vitamin C, xitrat va oxalat Irong mau nudc lieu Irong Ihyc le.
Idp lai cmi phirang phdp PCR
M a u
2
M S u 1 2 3 4 5 6
Vita innn Ham lugng T i t 10''(niol/l)(n-5)
1,30 1.27 Bang 3 Ki'.
aJiil IiK/iicn III a
Vitamin C
Xitrat
Oxalat C
RSD
(%)
10.37 10.63 t qua xdc ilinh di
'^dllji do chili tlicmihii.in
0 . 5 , 1 0 ' M 0.8.10"M 1.20,10"M O.S.IO'M 0 . 8 0 , I O ' M 2 . 5 . 1 0 - ' M
Hill lO'l
i di'ing d(i Ihh
Xitrat in luong TB [inol/l)(n=5)
1.98
RSI)
(%)
7,69 2.14 5.39 cua phtrong phdp PCR hdi vitamin C. vtlral
\dng do chdt triri'rc Uii Ihem chtiih,
1.24.10-'\1
1.66,10"M
1 , 2 4 , 1 0 " ' M
Oxaial Hilm lu'ong TB I0"'(mol/ll(n=5)
1.23 1.06 tlidng qua them ehudn
\dng do chdt Mill Do kill them chudn
1 . 3 4 . 1 0 " ' M 1.58.10""M 2.69 10"M 2.30,10""M 2,04. I O ' M 3.56. l O ' M
RSD
(%)
1,77 1.41
thu hoi
(%)
79,0 77,5 94,1 93,5 100.0 95.2
Tr^n Ou6c To^n va Dig Tap chl KHOA HtJC & CONG NGHE 106(06)'3-8
STT 1 2 3 4 5 6 7 8 9
Bang 4 Ten benh nhSn
A A B C C D D D D
Ham Immg cdc chat Thoi diem
lay mau Sang Trua Chieu Sang Sang Trua Trira Trira Trira
ph. :/;j tich Irong mdu thin Tong the lich nu'6e
lieu lav (1 lan) 200 ml 200 ml 200 ml 200 ml 200 ml 200 ml 200 ml 200 ml 200 ml
,' lc Iheo phi C^„„, 10"'(M)
3.02 3.41 9.27 4.06 4.42 2.28 2.26 2.22 1.92
long phdp I^C'R C ,„
lO'(M) 2.04 0.21 2.80 0.17 2.40 1.04 1.20 1.06 1,23
Cv 10''(M)
1.20 1.56 1.33 1.72 3.43 1.11 1.06 1.35 1.36 KET LUAN
Trong qua trinh nghien ciiu xac dinh ddng llidi vitamin C, xilrat va oxalat trong mau nude lieu ma khdng can phai lach loai chimg ra khdi nhau chiing tdi da xiie dinh dieu kien toi uu cho phep do quang \ a md hinh PCR de phan tich cac mau thuc Ie. Phuang phap lidi qui cau tu chinh (PCR) sir dyng phan mem Matlab xac djnh dong thdi vitamin C, xitrat va oxaial trong mau nude tieu cho ket qua nhanh chong ma khong phai tach toai va lam giau.
TAI LIEU THAM KHAO
[1]. D6 Dinh H 6 (2007). So toy xel nghiem hod iinh lain sdng. Nxb Y hoc. Ha Noi
[2]. ^1ci^s;lln Moroozifar. Mozhgan Khorasani - Motlagh (2003), " Applicalion of Poia-'isium Chromate - Diphenylcarbazide in the qitanliiaiion delernimalion of ascorbic acid hy Speciropholomeir) ". Turk.J.Chem 27, 717- 722.
[3], Maciej Rutkowski, Krzysztof Grzegorczyk and Janusz Greger (2002),"Adaptation o] the phospholungslale method jor ihe delerminalion of
I 'ilaiiiin C contents in animal and human li.y'iiie.s", Z NaturforschC Nov-Dcc. 57(11-12), 1062- 1065 [4] Ma,Yong|un; Zhou, men, Jin, Xiaoyong (2002), '''Flow-inflection chemiliiininescence delerminalion of ascorbic acid In' inc of Cerium ill') - Rhodamin B syslem". Analylica Chimica Acta. 464 (2). 2 8 9 - 193
[5]. Sura C.Cunha, Isabel M.P.L.V.O.Ferrira. Jose.
O Fernades, Miguel A, Faria, P.P.OMveira and M.A.Ferrira (2001), Delerminalion of Lactic . acetic, succinic, and citric acid in table olives by HPLC/UV- J LIO CHROM and /?c'/.Techno-24(7), 1029-1038.
[6] Salah M.Sulian, Anthony D, Walmsley (1997), "Simidlaneous Kincite Mellvid for the Delerminalion of Vitamin C. Citraie and Oxalate Employing the Kalman Filler". Analyst . Vol 122(1601-1604).
[7] Chen ZG. En BT. Tiuo-ng Zq (2002).
Siimdlaneoui and direct delerminalion oJ oxalic acid, larlanc acid, malic acul, vilamin C. citric acid, and succinic acid in Frucliis mume by reversed- phase high-performance liquid chromatography, J Chromatogr Sci. Jan, 40(1):
35-39.
Tran Quoc Toan vd Dig Tap chf KHOA UQC & CONG NGHE
SUMMARY
S I M l i ; ! ANKOUS DEI ERMENATION OF VITAMIN C, CITRATE AND OXAJ.ATE IN THE URINE USINC KINETIC SIM f TROI'IIOTOMETRIC METHOD
Tran Quoc Toan , Mai Xuan Truong Collfgi- ofFducaUon - TMJ Tills .study investigaics the the research results for siiuullaneous determination of vitamin C, citrate and n\,ilatc in unne by kinetic pliotometric mclliod using Ce (IV) as oxidants
E\pcriiiicnls measuring the absorbance ol'thc .solution was carried oul after 20 seconds from when adding Cc (IV) at a \\jvclength of 32Onm, compared with the H7SO1 0,7M in the measuring period 20-120 seconds the absorbance data were proccsscdusing MATLAB software based on niiillivaiuiic regression equations were established. The experimental results showed that ihe detection limit vitamin C, cilrale and oxalaie respectively 7,75,I0-7M. 7,38.I0-7M and 7,17.10- 6M; which restrict the iunount of vitamin C, cilrale and oxalate respectively is 2.5 I0-6M, 2.46.IO-6M\;i2.39,IO-5M.
Key wordfi: kinelic.•.peclrophotomelric. viluniinC. mrale. oxalate. ceriuiiilIT/. urine
Ngdy nluin hdi. 11/6/2013. \gdy phan hien- 12/6/2013, Xgiiy diiyei ddng.26/7/2013
•Icl 0'rs55.i'jn.s Ijuail qiKKioankS/lS'dgmaiict
