1.

Khi nim
Phng php sc k lng hiu nng cao (HPLC) ra i trn c s pht trin v ci tin t phng
php sc k ct c in. HPLC 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 bng lin kt ha hc vi cc nhm chc hu c.
Phng php ny ngy cng c s dng rng ri v ph bin v nhiu l do: c nhy cao, kh
nng nh lng tt, thch hp tch cc hp cht kh bay hi hoc d phn hy nhit.
Phm vi ng dng ca phng php HPLC rt rng, nh phn tch cc hp cht thuc tr su, thuc
khng sinh, cc cht ph gia thc phm trong lnh vc thc phm, dc phm, mi trng
2. Phn loi
Da vo s khc nhau v c ch tch chit s dng trong HPLC, ngi ta chia HPLC thnh 4 loi

Sc k hp ph hay sc k lng rn (adsorption/liquid chromatography).

Sc k phn b (partition chromatography).
Sc k ion (ion chromatography).
Sc k ry phn t (size exclusion/gel permeation chromatography).
3. Pha tnh trong sc ky pha ao
Trong sc k phn b ni chung, pha tnh l nhng hp cht hu c c gn ln cht mang rn silica
hoc cu thnh t silica theo hai kiu:

Pha tnh c gi li trn cht mang rn bng c ch hp ph vt l sc k lng-lng (liquid-liquid

chromatography).
Pha tnh lin kt ha hc vi cht nn sc k pha lin kt (bonded phase chromatography)
Trong qu trnh s dng, ngi ta nhn thy sc k pha lin kt c nhiu u im hn sc k pha lnglng v mt s nguyn nhn sau:
o
o

Pha tnh trong h sc k lng-lng d b ha tan bi pha ng nn d b mt mt pha tnh trong

thi gian s dng v gy nhim i vi hp cht phn tch.
Do pha tnh ca sc k lng-lng d tan trong pha ng nn ngi ta khng th ng dng
phng php ra gii gradient dung mi.

V vy, ngi ta thng ch quan tm n loi sc k phn b pha lin kt v phn ln cc loi ct s dng hin
nay trong sc k phn b u c cu trc dng ny.
B mt cc ht silica SiO2 (cc ht ny c ng knh 3, 5 hoc 10 m) c x l (thy phn) bng cch un
nng vi HCl 0,1M trong mt hoc hai ngy to ra nhng nhm SiOH nh sau (thng thng ch c khong
8 mol SiOH/m2 b mt):

Hnh 1. B mt silica thy phn

Sau b mt silica thy phn ny s c cho phn ng vi cc organochlorosilan to ra cc pha tnh
khng phn cc, phn cc trung bnh hoc rt phn cc ty theo nhm R gn vo.

Hnh 2: To nhnh trn b mt silica

Thng ch khong 50% nhm OH mt H+ to ra HCl (tc < 4mol/m2 b mt b silan ha) v s kt hp s
dn dn b nh hng bi hiu ng lp th. Ngoi nhm Cl ngi ta cn s dng OCH3.
Hp cht cn phn tch khi i qua pha tnh s b gi li bi nhng lc lng tng tc khc nhau ty thuc
tnh cht, c im ca cht tan v pha tnh.
Trong SKP, nhm th R trong hp cht siloxan hu nh khng phn cc hoc t phn cc. l cc ankyl
dy di nh C8 (n-octyl), C18 (n-octadecyl) cn gi l ODS (octadecylsilan) hoc cc nhm alkyl ngn hn nh
C2; ngoi ra cn c cyclohexyl, phenyl trong nhm phenyl c phn cc cao hn nhm alkyl. Ngi ta
nhn thy cc alkyl dy di cho kt qu tch n nh hn cc loi khc nn y l loi c s dng nhiu
nht.

Hnh 3: Cu trc ca ct ODS

Tuy nhin do hiu ng lp th nn trong cu trc ca pha tnh cn nhm OH cha phn ng, gy nh hng
xu n qu trnh tch sc k ty mi trng pH phn tch.
Trong mi trng qu acid (pH < 2) th c s phn ly cc nhm ether (-O-Si-C18) ra khi nn. Lc ny ct s
mt hot tnh dn n cht cn phn tch khng cn tng tc tt vi pha tnh na.
Trong mi trng baz (pH > 7), chnh nn silic mang pha tnh c th b ha tan (SiO2 thnh silicat), h qu l
N gim v s nhnh ghp cng gim, mi rng ra v thi gian lu cng c th gim. Kt qu phn tch nh
vy s mt i chnh xc.
Mt trong nhng cch khc phc hin tng ny l dng cc cht nh trimethylchlorosilan ClSi(CH3)3 hoc
hexamethyldisilazan (t s dng hn) tng tc vi nhm OH ny (gi l hin tng end-capping). Lc ny
ta s c loi ct t tng tc vi cht phn tch c tnh baz (ct LC-DB ca hng SUPELCO).

Hnh 4. Cu trc ct LC-DB

C mt cch khc loi tr bt nh hng ca nhm OH m khng cn tng tc vi n l thay nhng
nhm methyl ca Si(CH3)2-C18 bng nhng nhm th ln hn nh isopropyl nhng nhm ny s che i
nhng nhm OH, cn tr tng tc ca nhm OH vi cht cn phn tch.

Hnh 5: Cu trc ct c gc isopropyl

Ngi ta cn ghp ln dy C18 mt s nhm phn cc tng thm phn cc ca dy C18, lm ct c kh
nng tch chn lc hn i vi nhng hp cht phn cc mnh (ct EPS Expended Polar Selectivity).
Ngoi sn silica, thi gian gn y ngi ta c s dng n nn nha polystyren (Polystyren Reversed Phase
PRP) cho php phn tch trong mi trng pH t 1 13. Ct ny d s dng trong mi trng acid v baz
mnh.
4. Pha ng trong sc k pha o
Pha ng trong sc k lng ni chung phi t nhng yu cu sau:
Ha tan mu phn tch.
Ph hp vi u d.
Khng ha tan hay lm mn pha tnh.
C nht thp trnh p sut di li cao.
Tinh khit dng cho sc k (HPLC grade).

Bng 1: Tnh cht ca mt s pha ng trong sc k lng

Hn hp nc v dung mi hu c thng lm gia tng nht dn n vic tng p sut ct.

Hnh 6: nht ca hn hp nc v dung mi hu c 25oC

Vic la chn dung mi v thnh phn dung mi trong pha ng c ti u ha cho nhng hp cht cn phn
tch. Thng thng, ngi ta s dng hn hp dung mi MeOH/nc trc, ri ACN/nc hay THF/nc. Vi
mt hn hp cht phn tch phc tp th s c s trn ln ca cc dung mi hu c vi nc.
Thnh phn pha ng c th c nh trong sut qu trnh chy sc k (ch isocratic) hoc c thay i theo
mt chng trnh nh sn (chng trnh gradien dung mi) c hiu qu tch tt hn.
detector .
. Detector c s dng rng ri nht l detector ph khi . Loi ny nhy nh detector bt electron hoc
detector hunh quang. Detector dn nhit l cch ph bin nht d tm cc cht nhng n khng nhy
cho nhng ct mao qun m c phn gii cao.nn khi cn bit nhng thng tin nh tnh nhn din cc
cht ra gii, cc detector khi ph v hng ngoi l nhng chn la tt.