Bo Y Te Vien Kiem Nghiem Thuoc TP.

Ho Ch Minh

KY THUAT QUANG PHO T NGOAI-KHA KIEN UV-Vis THIET B VA HIEU CHUAN

DAN BAI
I.
1.1. Cac thanh phan 1.1.1. en nguon 1.1.2. Bo phan tao anh sang n sac 1.1.3. Cong o va ngan cha cong o 1.1.4. Detector 1.1.5. He thong quang hoc

THIET B

1.2. May quang pho thong thng 1.3. May quang pho diode array

1.4. Cau hnh

1.4.1. Kieu mot chum tia 1.4.2. Kieu hai chum tia 1.4.3. Kieu Split-beam 1.4.4. Kieu Dual-wavelength

II.

CAC VAN E KY THUAT THNG GAP VA BIEN PHAP KHAC PHUC TRONG VIEC NG DUNG PHNG PHAP QUANG PHO T NGOAI KHA KIEN VAO KIEM NGHIEM THUOC

III. HIEU CHUAN IV. VAN HANH

Mot so khai niem c ban

Ban chat cua anh sang
Tnh chat song:
Bc xa co dao ong song cua cng o ien trng va t trng thang goc nhau. Anh sang nhn thay, tia hong ngoai, tia t ngoai. eu la cac dang khac nhau cua bc xa ien t, ch khac nhau ve o dai song (bc song)

Thh chat hat

Bc song (): khoang cach ngan nhat gia 2 dao ong cung pha. n v nm = 10-9 m.

Bc xa ien t la nhng hat mang nang lng goi la photon lan truyen vi van toc anh sang E = hc/

Mot so khai niem c ban

Bc xa vung UV-Vis (tia t ngoai, anh sang nhn thay) chia thanh 3 vung nho:
Vung t ngoai chan khong (UV xa): = 50-200 nm t s dung trong kiem nghiem v: - Co nang lng kha ln, khi va cham gay v lien ket trong phan t - B hap thu manh bi hau het dung moi va oxy cua khong kh - B hap thu bi thach anh (dung lam coc o)

Vung t ngoai gan (UV gan): = 200-340 nm

Vung kha kien (Vis): = 340-800 nm

THIET B
May quang pho t ngoai kha kien UV-Vis

CAC THANH PHAN CHNH CUA MAY UV-Vis

en nguon (1) Bo phan tao anh sang n sac (2) Cong o va ngan cha cong o (3) Detector (4) He thong quang hoc (5)

EN NGUON (LAMP) (1)

AC IEM CUA NGUON SANG LY TNG
Cng o sang hau nh khong thay oi va u manh moi bc song trong vung t ngoai kha kien. o nhieu thap

Can dung on ap e on nh cng o bc xa cua en nguon

EN DUNG CHO VUNG T NGOAI

en Deuterium
Deuterium b kch thch ien ap suat thap tao ra pho phat xa lien tuc trong vung t ngoai Bong en lam bang thach anh v thuy tinh hap thu manh anh sang vung bc song di 350 nm Cho cng o sang at yeu cau s dung lien tuc trong vung t ngoai (t 200 en 340 nm)

Cho cc ai bc xa hu ch trong vung kha kien (t 656,0 en 656,2 nm) c dung e kiem tra o ung cua bc song may quang pho. Nhieu gay han che en hieu nang cua may. Thi gian ban huy (cng o en giam ch con phan na so vi gia tr luc mi lap at) xap x 1.000 gi.

EN DUNG CHO VUNG KHA KIEN

en Tungsten
Si ot lam bang wolfram. Chat lieu lam bong bang thuy tinh. Nhiet o si ot hoat ong la 2.870 K.

en Tungsten/Halogen
Co mot lng nho iod ben trong vo en bang thach anh cha si ot wolfram. Nhiet o si ot hoat ong gan 3.500 K.

 S phan bo nang lng phu thuoc vao nhiet o. vung kha kien, nang lng en thay oi theo ien the hoat ong. Do vay can thiet dung on ap.

ac iem

Cho cng o sang at yeu cau s dung tren mot phan vung t ngoai va toan bo vung kha kien (t 340 en 900 hay 1.100 nm). o nhieu rat thap, rat t troi ng nen. Thi gian s dung khoang 10.000 gi.

SO SANH EN TUNGSTEN VA EN TUNGSTEN/HALOGEN

Tuoi tho cua en tungsten/halogen cao hn gap oi so vi en tungsten.

en Tungsten, tungsten b thang hoa di tac dung cua
nhiet o cao lam si ot b mon dan theo thi gian va b t e lai vet en tren au bong en.

 Ket qua la tuoi tho en b giam. en Tungsten-Halogen, iod phan ng vi hi tungsten b thang di tac dung cua nhiet o cao hp chat WI2. Chat nay (WI2) ngng tu tren ot giai phong wolfram, nen khong b mon, gia tang tuoi

tao

hoa thanh
si ot

tho en.

si

EN XENON
Tao ra nhng bc xa co cng o ln bang cach cho dong ien iqua bau kh xenon.

ac iem

Cho pho lien tuc t 250 - 600 nm.

Cho cng o sang at yeu cau lien tuc trong vung UV-Vis.

Thng dung trong nhng ng dung nh o t le tan xa pho huynh quang can cng o cao.

hay

quang

 Cac thanh phan quang hoc cua mot bo phan tao anh sang n sac bao gom: khe vao (1), knh chuan truc (2), bo phan tan sac gom lang knh hay cach t (3), mat phang hoi tu (4), khe ra (5). Tao ra anh sang vi cac bc song khac nhau c tan sac nhng goc khac nhau.

BO PHAN TAO ANH SANG N SAC (MONOCHROMATOR) (2)

 Bo phan nay dung e chon t nguon sang mot anh sang n sac co bc song mong muon i qua mot khe ra thch hp. Bo phan tan sac: co 2 loai thong dung la lang knh va cach t.

LANG KNH (PRISM)

ac iem

Tao ra anh sang nhieu mau sac nh anh sang mat tri.

cau vong t

Cau tao n gian va re tien nhng cho goc tan xa khong tuyen tnh va phu thuoc nhiet o.

Nhc iem cua lang knh

Pho cho bi lang knh khong tuyen tnh ve bc song. Lang knh hap thu mot phan nang lng anh sang khi i qua no. Goc tan xa phu thuoc nhiet o. Phai tao ra c che quay lang knh tng oi phc tap e co the kiem tra bc song t thang o.

CACH T (GRATING)
S tan sac cua bc xa t ngoai kha kien co the c tao ra bang cach hng mot chum tia a sac cho i qua cach t dan truyen hoac ve pha cach t phan xa (dung nhieu hn trong thc te). Cach t phan xa la mot ban thuy tinh ma nhom. Tren be mat ma nhom nay co nhng ranh song song va gan ke nhau.

 Phan b rach khong phan chieu anh sang, phan con lai ong vai tro mot gng phang gom nhieu gng rieng biet nhau. Anh sang chieu ti phan ma nhom khong b rach nay se b phan xa tr lai theo nhng goc khac nhau tuy thuoc vao bc song cua chung.
Cac tia phan xa se giao thoa vi nhau tao nen van nhieu xa co bc song khac nhau.

 Khi quay cach t, ta thu c khe

van co bc song khac nhau.

ra

cac

Cach t phan xa anh sang nhng bac khac nhau nen phai dung knh loc e ch cho anh sang cua bac anh sang phan xa mong muon en au do.

CACH T LOM (CONCAVE GRATINGS)

c tao thanh tren be mat lom Cho phep thiet ke mot bo tao anh sang n sac ma khong can bo phan chuan truc phu tr, gng hoi tu v be mat lom co the phan tan roi va tap trung bc xa khe ra.

Loai nay co u iem la gia re, s giam thieu cac be mat quang hoc lam gia tang mc nang lng cua bo tao anh sang n sac cha cach t lom.

HOLOGRAPHIC GRATINGS
Ky thuat quang hoc tao cach t tren be mat thuy tinh phang hay lom bang tia laser. Ky thuat nay cho phep tao cac ranh ong eu ve kch thc va hnh dang. Cach t loai nay cho cac pho khong co anh sang lac va nhieu (ghost).

 Tao thanh bang cach chieu 2 chum tia laser giong nhau t 1 goc thch hp len mot be mat thuy tinh co tam chat can quang.
Van giao thoa tao thanh t 2 chum tia tren tang nhay chat can quang lam cho no tan ra e lai mot cau truc co khac vach Sau o phan con lai c ma nhom hoac nhng chat phan xa khac e tao thanh cach t phan xa

U IEM CUA CACH T SO VI LANG KNH

Cho o phan giai tot hn

Cho s tan sac tuyen tnh

o rong cua giai pho la hang tnh C che chon bc song n gian hn

CONG O VA NGAN CHA CONG O (CELL & CELL HOLDER) (3) CONG O

c lam bang chat lieu sao cho anh sang trong vung o phai truyen qua (trong thc te cac loai cong o co o truyen toi a la khoang 90% moi bc song trong vung UV-Vis) Trong vung t ngoai (di 350 nm) dung dung coc lam bang thach anh hay silica (silic dioxid) nong chay.

 Trong vung kha kien dung dung cong lam bang thuy tinh silicat hoac bang chat deo (nha acrilic). Quang lo cong o thong thng nhat la 1 cm.

Ngoai ra con co cac cong o co quang lo t 0,1 en 10 cm dung cho cac nong o thch hp ma khong can pha loang tiep theo.

KIEU CONG O TIEU CHUAN

Pho bien nhat la loai cong o hnh khoi ch nhat. Quang lo coc o thong thng nhat la 1 cm. Ngoai ra con co cac cong o co quang lo t 0,1 en 10 cm e dung cho cac nong o thch hp ma khong can pha loang tiep theo. Neu the tch mau co gii han th dung cong o co ca so.

MOT SO KIEU CONG O AC BIET

Microcell La loai cong o co ca so rat nho (co mat cat 2 x 2,5 mm). Ch can lng mau xap x 60 l e o. Ultra-microcell La loai dung cu o mi thiet ke e o the tch mau c microlit (2-5l) bao gom mot bo phan tiep hp va mot ong mao quan bang silica nong chay quang lo ngan dung mot lan.

 Cho trc tiep mau o vao ong mao quan, sau o cai vao bo phan tiep hp. Quang lo giam cho phep o cac mau co nong o cao ma khong can pha loang. Ap dung e o nong o va o tinh khiet cua AND va nong o protein.

Coc o kieu dong chay lien tuc

Dung trong trng hp t ong hoa nh trong may quang pho ghep noi vi may o o hoa tan.

NGAN CHA CONG O (CELL HOLDER)

Ngan cha cong o e o o uc (Turbidity Cell Holder)

Ngan cha cong o c dch chuyen ra xa detector so vi v tr bnh thng va mot khe at gia mau va detector. Khe nay ch cho phep anh sang lo i qua mau ma khong cho anh sang tan xa en en detector.

Micro-Auto 12 Cell Holder

La loai ngan cha cong o dung trong sinh hoa e phan tch ong hoc enzym. Bao gom cac ngan cha cong o co the tch toi thieu 100 l va bo phan ieu hoa nhiet o bang dong nc hoac kieu Peltier.

Water circulated Cell Holder

La loai ngan cha cong o duy tr c nhiet o bang mot dong nc luan chuyen lay t may ieu nhiet (Thermostat). Dung trong phan tch enzym.

Micro-Auto 6 Tm Cell Holder

La loai ngan cha coc o dung trong sinh hoa e xac nh tm (thermal melting point) cua AND. Bao gom mot bo phan kiem soat nhiet o kieu Peltier, quat lam nguoi ngan cha coc o bang khong kh, bo phan lam sach hi ngng tu tren thanh coc o dung kh nitrogen, hai ien cc hieu nang cao Peltier, bo phan o nhiet o bang cam bien nhiet.

DETECTOR (4)
Detector te bao quang (Phototube)
La cac ong thuy tinh hoac silica c tao chan khong vi cathod la kim loai kiem, anod tch ien dng.

Khi anh sang chieu ti cathod, cac electron c tach ra va i ti anod lam cho the tang len t le vi cng o anh sang.

o nhay pho cua cac te bao xac nh bang chat lieu cathod, vi antimon/cesium t 190 600 nm, vo oxid cesium/bac t 600 1000 nm.

Detector ong nhan quang (Photomultiplier)

Nguyen tac hoat ong gan giong te bao quang nhng co them bo khuech ai ben trong lam cho loai nay co o nhay ln hn tren thang pho rong hn. Anh sang gay ra s bc xa photon t photocathod, giong nh trong

te bao quang nhng anod c sap xep thanh day dynod co the tang len khong ngng.

Detector silicon diod (Silicon diode)

Co hieu nang cho toan bo thang o rong, vng chac, o tho cao hn ong nhan quang va khong tieu hao nhieu ien nang.

Chuoi diod la mot tap hp cac detector photodiod rieng biet xep at ke nhau tren tinh the silicon tuyen tnh trong ma tran tao nen.
Moi diod co mot tu ien va c noi vi dong au ra bang mot cai ngat.

 au tien tu ien c tch mot gia tr ien nhat nh.

Khi photon tham nhap vao silicon, nhng phan mang ien tch t do tao thanh e xa tu ien.
Tu ien lai c tch ien nhng chu ky ac trng cho chu ky o oi vi moi chu ky quet.

 Lng ien can e nap cho tu ien t le vi so pho ton c phat hien bi moi diod hay noi cach khac t le vi cng o sang. Pho hap thu thu c bang cach o lng s thay oi cng o sang tren toan khoang bc song. Thong thng detector chuoi photodiod cha 1024 detector photodiod rieng biet.

HE THONG QUANG HOC (OPTICAL SYSTEM) (5)

S dung cac thau knh hay gng lom e tap trung anh sang. Neu dung loai knh thong thng co the b sai lac ve mau sac.
Knh tieu sac ket hp nhieu loai knh vi ch so khuc xa khac nhau khong gay ra hien tng sai lac ve mau sac nhng gia thanh cao.

 Gng lom re hn knh tieu sac va cung khong b sai lac ve mau sac nhng be mat nhom de b an mon lam giam hieu nang. Tren moi be mat quang hoc ke ca giao dien gia cac thanh phan cua knh tieu sac, 5 10 % anh sang the hien qua s hap thu hay phan chieu. Mot may quang pho ly tng phai thiet ke vi be mat quang hoc toi thieu.

MAY QUANG PHO THONG THNG

Anh sang a sac t en nguon tap trung khe vao cua bo tao anh sang n sac cho truyen qua mot cach chon loc mot day anh sang hep. Day anh sang nay i qua mau, lo ra va en detector. o hap thu cua mau la hieu gia cng o anh sang ti detector cua mau trang va mau th.

 May loai nay thch hp e o o hap thu mot bc song tren pho nhng khong thch hp lam khi o nhng chat khac nhau nhng bc song khac nhau hoac khi lay pho cua mot chat. e lam c ieu nay, bo tao anh sang n sac phai xoay mot cach c hoc gay ra sai so do o tai lap. Qua trnh thu nhan d lieu can co thi gian.

MAY QUANG PHO DIODE ARRAY

Anh sang a sac t en nguon i qua mau roi tap trung khe vao cua bo tao anh sang a sac sau o b tan sac tren day diode, moi diode o mot day hep cua ca pho. Kch thc cua diode va khe vao cua bo tao anh sang a sac co lien he en o rong day cua anh sang phat hien.

 Moi diode thc hien chc nang nh mot khe ra cua bo tao anh sang n sac. e giam thieu phan ng quang hoa, may co ca chap e chan anh sang t en nguon cho ti tien hanh o mau. May loai nay cho phep thu nhan d lieu rat nhanh, o tai lap bc song rat tot va o tin cay rat cao.

u iem
Ghi pho nhanh Dung e nghien cu ong hoc nh tnh va nh lng cac thanh phan tach ra t cot sac ky

Nhc iem
Nang suat phan giai kem (1-2nm) Gia thanh cao

CAU HNH
Kieu mot chum tia
Co the la loai may quang pho hoac may quang pho day diode. thong thng

He thong quang hoc n gian nen co o nhay cao va gia thanh ha.

 May quang pho oi chng do Vien tieu chuan va ky thuat quoc gia Hoa ky va phong th nghiem vat ly quoc gia Anh quoc s dung la loai may mot chum tia.
May quang pho day diode ac biet thch hp cho cau hnh mot chum tia v thu c pho rat nhanh va khoang thi gian o gia mau trang va mau th b giam en mc toi thieu.

Kieu hai chum tia

Chum tia i t en nguon ti bo tao anh sang n sac c tach ra thanh hai chum tia n sac bang nhau do mot bo chan sang t ong xoay vai giay mot phut (chopper), mot chieu ti mau o, mot chieu ti mau oi chieu

Kieu split-beam
Giong vi loai may 2 chum tia nhng dung bo tach chum tia (beam splitter) thay v chopper e cho anh sang i qua mau trang va mau th ong thi e en 2 detector rieng biet giong nhau.

Vi cau hnh nay, mau trang va mau th c o ong thi. He thong quang hoc cung n gian hn may 2 chum tia thc thu. Nhc iem la hai detector co the khong hoan toan ong nhat nhau.

Kieu dual-wavelength
Cau hnh nay cho phep o 2 bc song ong thi nh khi nghien cu 2 phan ng xay ra ong thi ben trong mau.

Bo tao anh sang n sac cha 2 bo phan tan sac. May loai nay at hn may quang pho thong dung va c dan thay the bang may quang pho diode array co the ghi pho ong thi nhieu bc song khac nhau.

Bo Y Te Vien Kiem Nghiem Thuoc TP.Ho Ch Minh

CAC VAN E KY THUAT THNG GAP VA BIEN PHAP KHAC PHUC TRONG VIEC NG DUNG PHNG PHAP QUANG PHO T NGOAI KHA KIEN VAO KIEM NGHIEM THUOC

EN KHONG CHAY KHI M NUT POWER

Nguyen nhan
1a. Day nguon khong noi vi may 1b. No cau ch nut power 1c. Chay en nguon 1d. Mach ien b hong 1e. The nguon thap

Khac phuc

1a. Kiem tra day nguon 1b. Thay cau ch nut power 1c. Thay en 1d. Goi chuyen vien bao tr 1e. Dung on ap

GIA TR O HAP THU CAO BAT THNG VA KHONG TR VE 0 KHI NHAN PHM AUTO ZERO
Nguyen nhan
Mot trong 2 hoac ca 2 en nguon b chay hoac khong m

Khac phuc
M en len. Thay en

O HAP THU DAO ONG MANH

Nguyen nhan
3a. Chum tia oi chieu b chan 3b. en tat hoac b chay

Khac phuc
3a. Kiem trang i cua chum tia 3b. Kiem tra nut bat en va tnh trang en

O PHANG NG NEN NAM NGOAI GII HAN CHO PHEP

Nguyen nhan
Chum tia i qua mau b chan mot phan

Khac phuc
Don sach vung chum tia i qua mau va tien hanh hieu chnh ng nen

BC SONG HIEU CHNH NAM NGOAI GII HAN CHO PHEP

Nguyen nhan
H hong ve c hay ve ien

Khac phuc
Goi chuyen vien bao tr

NHIEU NG NEN LN BAT THNG

Nguyen nhan
6a. o nhay detector kem 6b. Mot trong nhng bo phan quang hoc sau ay b h hong: Bo phan chuyen oi nguon sang Khe sang Knh loc

Khac phuc
6a. Goi chuyen vien bao tr 6b. Goi chuyen vien bao tr

NHIEU NG NEN LN BAT THNG

Nguyen nhan
6c. en nguon a het han s dung 6d. ien the cung cap cho thiet b thap hn quy nh 6e. Nhieu co nguon goc ngoai lai

Khac phuc
6c. Thay en nguon 6d. S dung on ap 6e. at thiet b xa cac trng ien t hoac cac nguon gay rung lac

NHIEU NG NEN LN BAT THNG

Nguyen nhan
6f. Thiet b khong noi at 6g. o rong khe sang nho

Khac phuc
6f. Noi at cho thiet b 6g. Tang gia tr o rong khe sang

O LECH D LIEU LN KHONG CHAP NHAN C

Nguyen nhan Khac phuc
7a. Thay en nguon 7a. en nguon b yeu 7b. ng i cua chum tia b anh hng do dau van tay hay bui 7c. Cong o hay ca so b ban

7b. Ra cong o hay/va ca so 7c. Ra cong o hay/va ca so

MAY KHONG OC C TAT CA CAC NH THEO QUY NH

Th du: Rifampicin co 4 cc ai hap thu 237, 254, 334 va 475nm. May khong ghi c nh 237nm

Nguyen nhan
at o rong khe sang (slit width) khong phu hp

Khac phuc
Giam o rong khe sang

O HAP THU DAO ONG MANH

Nguyen nhan
Dung cong o thuy tinh e o mau bc song t ngoai

Khac phuc
Thay bang cong o thach anh

O HAP THU GIAM DAN

Nguyen nhan
Dung moi b boc hi Bot kh hien dien tren thanh cong o khi cho dung dch vao

Khac phuc

Dung cong o co nap ay

Lam ay cong bang pipet

O HAP THU THAY OI ANG KE GIA CAC LAN O

Nguyen nhan
Dung cac cong o co o thau quang khac nhau Khong nh hang cong o

Khac phuc

Dung mot cong o e

o cac mau

S dung cong vi cung mot mat quay ve pha anh sang

NH HAP THU B LECH VE PHA BC SONG NGAN

Nguyen nhan
Hien tng Tracking (Tracking error) do quet pho toc o qua nhanh oi vi may hai chum tia

Khac phuc

Giam toc o quet pho

Ghi pho chuan trong cung ieu kien

NH HAP THU B THAY OI SO VI GIA TR QUY NH

Nguyen nhan
Dung moi s dung khong chuyen dung cho quang pho Dung moi co lan nc

Khac phuc
Dung loai dung moi thch hp

Hieu chuan May quang pho UV-Vis

MUC CH
Xac nh o on nh, o ung, o chnh xac cua may. Kiem tra tnh trang hoat ong cua may Phat hien nhng khiem khuyet cua thiet b Cung cap cac d lieu cho ngi bao tr e xac nh nhng hong hoc cua thiet b nham ho tr trong sa cha

NOI DUNG

Kiem tra o phang ng nen (check of baseline flatness)

Tren toan thang bc song, o thang giang (Fluctuation) cua ng nen so vi khong kh phai nam trong khoang qui nh cua bang thong so ky thuat.

Th du - May Hitachi U3210 co o thang giang khoang 0,001Abs - May Hitachi U2010 co o thang giang khoang 0,002 Abs (200-950nm) - May Hitachi U3000 co o thang giang khoang 0,001Abs

trong trong trong

- May Ultrospec III co o thang giang trong Abs

khoang 0,002

Cong o ( cells or cuves )

Thng dung cac cong o tieu chuan Cac cong o phai am bao cac yeu cau sau: Phan be mat hng chum tia va cho chum tia i qua phai that phang, nhan va song song vi nhau. Hai ban anh sang ti vi anh sang i phai song song vi nhau. qua

 Quang lo trong cong o phai am bao gia tr danh nh Cong o thng c lam bang: Thach anh (Quartz) cho vung o t ngoai va kha kien Thuy tinh cho vung o kha kien Yeu cau cac cong o phai cho o hap thu hay o thau quang luon luon bang nhau khi cha cung mot dung moi hay cung mot dung dch o trong cong. Cac cong o co be day 10 mm co dung sai cho phep ve o dai quang trnh la 0,005 cm hay 0,05/10 = 0,005 = 5o/oo

Kiem tra bc song (control of wavelengths)

May phai am bao o ung cua bc song (wavelength accuracy) theo ch tieu ky thuat cua may do nha san xuat e ra va phu hp vi yeu cau cua cac Dc ien ( 1nm t 200 - 400nm; 3nm t 400 - 700nm). oi vi may quang pho UV-Vis the he mi, en D2 c s dung nh la mot nguon sang tao mot vach sang bc song 656,1 nm e kiem tra o ung cua bc song (656,4 655,8 nm).

 oi vi ngi hieu chuan may th s dung knh chuan holmium oxide hay dung dch holmium perchlorat e kiem tra o ung cua bc song. Trong trng hp may o iem va khong co chc nang quet pho, tien hanh o o hap thu tai cac iem xung quanh cac cc ai e kiem tra.

Kiem tra o hap thu (control of absorbances )

Kiem tra o hap thu cho biet o ung cua may (Photometric Accuracy). May phai cho cac thong so ap ng vi cac thong so ky thuat e ra cho moi may va yeu cau cua cac Dc ien. Vi may at c o ung theo qui nh cho phep co the thc hien phep o tuyet oi. Dung dung dch kali dicromat. Gii han hap thu la 0, 01 AU

o phan giai cho phan tch nh tnh (resolution)

Ghi pho cua dung dch toluen (TT) trong hexan (TT) 0,02 % (tt/tt). Gia tr toi thieu cua ty so gia o hap thu cc ai 269 nm va o hap thu cc tieu 266 nm phai ln hn 1,5
Ghi chu: Theo BP 2005, ch tieu nay khong bat buoc.

ao ham bac hai (second derivative)

Vi may cho phep thc hien cac bien oi t pho thong thng sang pho ao ham bac mot, bac hai hay bac cao hn phai kiem tra moi tng quan gia ao ham bac hai va nong o cua chat can khao sat. Moi tng quan nay phai tuyen tnh. May phai lay c ao ham bac hai theo ung qui nh.

Gii han anh sang lac (limit of stray light)

Anh sang lac (stray light) la nguon goc gay sai so chnh cho may nen c qui nh rat chat che khi hieu chuan may. Yeu cau khi o la tia chieu ti mau o phai that s n sac. Khi chum tia chieu ti bo tao anh sang n sac e tao thanh cac tia n sac, co cac quang sai nen dan en viec tao ra cac tia la i kem vi tia n sac chieu ti mau do s cha hoan hao cua s tan sac

 Mau khong hap thu cac tia nay

Tiep theo cac tia nay lai chieu en detector, lam giam o hap thu cua nh can khao sat nhat la khi o vung nang lng thap gan 200 nm va dung dch co o hap thu cao. Anh sang lac cung gay ra s sai lech am cho nh luat Lambert Beer khi o thang o co o hap thu cao.

Anh sang lac cung tao ra khi buong o (sample compartment) khong kn.
Yeu cau may phai cho gii han cua anh sang lac trong pham vi cho phep.

Van hanh

May quang pho UV-Vis

o iem (nh lng) Quet pho(nh tnh)
o ong hoc

o iem (nh lng)

Bat may, si am (warming up). Chon bc song can o. Cho chum sang i qua mau trang. Hieu chnh cong o. at mau vao khoang o. oc gia tr o hap thu.

Cac iem can lu y

Lam ve sinh cong trc khi o. Khong dung cong b nt hoac xc. S dung cung mot cong cho chum tia oi chieu. Hng cung mot mat cua cong ve pha anh sang. Chat long trong cong ng mau th va mau oi chieu eu phai khong co bot kh va tieu phan.

Cac iem can lu y

Lau cong can than bang giay tham mem hoac vai khong x. Khong x tay vao be mat trong suot cua cong o. Dung moi s dung khong c co o hap thu anh sang trong vung bc song can o. Cong mau th phai c ra t nhat 3 lan bang dung dch mau th trc khi o.

Cac iem can lu y

Kiem tra cong e am bao s khac biet ve o hap thu khong phai do cong b ban. Khong o o hap thu khi co bot kh. Khong s dung cloroform bc song di 245 nm. Dung moi s dung phai lay t cung mot chai. Neu dung moi la nc, nen dung nc mi cat.

Cac iem can lu y

Nen bao quan dung moi hu c trong chai thuy tinh mau, khong ng trong chai nha. Oxy trong dung dch lam tang o hap thu neu dung moi la con bc song di 230 nm. Cac hp chat cacbonat hap thu manh bc song di 240 nm. Can o nhanh va ay nap cong o.

Quet pho (nh tnh)

Tien hanh giong nh o iem. bc song di 230 nm, o hap thu nen di 0,7. Nen quet pho UV t 370 nm en bc song ngan nhat van cho ket qua ung. Can at ung o rong khe sang e co o phan giai can thiet.

o ong hoc
Khao sat o ben cua mau va van toc cac phan ng thuy phan chat nen do enzym xuc tac. Tien hanh bc song xac nh.
Ghi lai s thay oi o hap thu theo thi gian.