CC QU TRNH TCH TRONG SC K LNG

TS NGUYN B HOI ANH

 Qu

Hiu qu ca qu trnh ny ph thuc rt nhiu Mc ch chnh ca sc k l tch v nh tnh

trnh tch l qu trnh quan trng nht trong phng php sc k ni chung v sc k lng ni ring.

vo tng tc gia cc cht trong pha tnh v pha ng.

cc cht trong hn hp cht phc tp Tuy nhin trong thc t khng phi lc no vic tch cc cht cng xy ra han ho do cc tng tc l rt phc tp v tng tc ca mi cht khi nm ring r v nm trong hn hp li phn no khc nhau
2

Mt s lai sc k
High

Performance Liquid chromatography (HPLC) : sc k lng hiu nng cao Gas Chromatography (GC) : sc k kh Thin-Layer Chromatography (TLC) : sc k lp mng Capillary Electrophoresis(CE) : sc k in di mao qun

u im ca phng php sc k
C

th phn tch ng thi nhiu hp cht phn gii cao nh qu trnh tch trn nhy cao (ppm-ppb) nh u d tch mu phn tch nh (1-100uL)
4

ct
Th

HPLC
High performance liquid chromatography High pressure liquid chromatography High price liquid chromatography High problem liquid chromatography High pleasure liquid chromatography
5

 Mt s dng HPLC
Pha thng (thun) : Normal phase Pha o : Reversed phase Ghp cp ion : Reversed phase ion pairing Sc k ion : Ion exchange (IC) Sc k gel : SEC (GPC/GFC) Sc k tch ng phn quang hc : Chiral separation Cch phn chia ny ph thuc vo ct tch

Phn chia HPLC theo cu to thit b

H ng dng (ng thnh phn pha ng) : Isocratic elution H thay i thnh phn pha ng : Gradient elution
6

Hn hp cht tch khi nhau th no ?

Hng dng chy

Pha tnh
7

Ti sao li c s khc nhau nh vy?

Mnh

Y u Do lc tng tc khc nhau

8

Qu trnh tch din ra th no

Qu trnh tch din ra trong ct sc k.

column
Vt liu nhi ct, 3-5um
9

Tng tc gia cht phn tch v vt liu nhi

Do s tng tc khc nhau gia cc cht phn tch v vt liu nhi ct m qu trnh tch xy ra.

sample A
packing material
sample B

10

Qu trnh tch
mixed sample
Mobile phase

column
11

u im ca HPLC
iu kin phn tch kh d dng :
- Khng cn bay hi mu nh GC, do phn tch c cc cht km bn nhit.

D dng thu hi cht phn tch vi tinh khit cao nu gn b thu hi phn on (fraction collector) , thng dng trong iu ch, tch tinh du, dc phm
lp li cao Thng khng phn hy mu
12

Sc k pha thun (thng) Normal Phase Mode

M. Tswett :

L ngi u tin pht trin phng php sc k, s dng tch Chlorophylls

Ber. Deut. Botan. Ges., 24, 384 (1906) Adsorptionsanalyse und Methode. Anwendung auf die Chemie des Chlorophylls.

chromatographische

13

Normal Phase
Petroleum ether

Chromatograph
Color

Chlorophyll's
CaCO3

14

Normal Phase
Trong h thng sc k u tin ngi ta s dng
CaCO3 lm cht nhi ct (pha tnh) Petroleum ether lm dung mi pha ng

V t pha thun c nh ngha nh sau :

Normal Phase
C t : Pha ng : mang tnh phn cc mang tnh khng phn cc
15

Cc loi ct HPLC pha thun

Ct Silica gel use) Ct Cyano use) Ct Amino Ct Diol

: dng a mc ch (general : dng a mc ch (general : phn tch ng : phn tch protein

-Si-CH2CH2CH2CN -Si-CH2CH2CH2NH2

Si Silica gel

Si

-Si-CH2CH2CH2OCH(OH)-CH2(OH)
16 Sillica gel bin tnh (b tr)

Cc lin kt xy ra th no?
Hydrogen bonding

Non-polar

Silica gel (polar)

17

Hydrogen bonding : Lin kt hydrogen

N u

cht phn tch c

: Nhm Carboxyl : Nhm Amino : Nhm Hydroxyl

-COOH -NH2 -OH

Lin kt Hydrogen s mnh.

N u

Do chng ngi lp th Lin kt Hydrogen s yu

mu c nhm tert-butyl hoc cc nhm khng phn cc ln

Retention Time v lin kt Hydrogen

Mnh SiOH SiOH Yu R t y u OH
2
1

HO

or

2
19

Dung mi pha ng s dng cho sc k lng pha thun

Dung mi ch yu (khng phn cc) :
Hydrocarbons (Pentane, Hexane, Heptane, Octane) Aromatic Hydrocarbons (Benzene, Toluene, Xylene) Methylene chloride Chloroform Carbon tetrachloride

Dung mi ph (phn cc hoc hi phn cc) :

Methyl-t-butyl ether (MTBE), Diethyl ether, Tetrahydrofuran (THF), Dioxane, Pyridine, Ethyl acetate, Acetonitrile, Acetone, 2-propaol, ethanol, methanol

Dung mi ch yu c s dng chnh lm pha ng, dung mi ph thng c thm vo vi t l nht nh thay i thi gian lu. Cc dung mi thng khng hp thu trong vng UV d dng cho vic xc nh. 20

nh hng ca phn cc
0 % MeOH 2 % MeOH 5% / MeOH

1 : Dioctyl phthalate 2 : Dibutyl phthalate 3 : Diethyl phthalate 4 : Dimethyl phthalate

Pha ng : Hexane
21

Pha o : Reversed Phase

Ct : Mang tnh khng phn cc Pha ng : Mang tnh phn cc
Normal phase
Ct : Pha ng : mang tnh phn cc mang tnh khng phn cc

22

Cc lai ct pha o
Ct C18 (ODS) Ct C8 (octyl) Ct C4 (butyl) Ct Phenyl Ct TMS

Non-polar property -Si-C18H37

Si

23

Cc lin kt xy ra th no?
Lin kt k nc
Dung mi phn cc

Khng phn cc
24

Tnh k nc
Nu mu c nhiu
CH3CH2CH2--- : dy Carbon : nhm thm (Aromatic)

Tnh k nc mnh hn.

Nu mu c nhiu :
-COOH -NH2 -OH : nhm Carboxyl : Nhm Amino : Nhm Hydroxyl

Tnh k nc s yu hn.
25

Thi gian lu v Tnh k nc

OH
1

C18 (ODS) Mnh OH

Yu
1 2

2
26

Cc dung mi s dng trong HPLC pha o

Dung dch m + dung mi hu c
Khi s dng dung dch m, nng m v pH l nhng thng s rt quan trng. Methanol (MeOH), acetonitrile (ACN) hay THF l cc dung mi thng s dng nht cho sc k pha o.

Vic ti u ha t l ca dung dch m v dung mi hu c cng rt quan trng. Vi cng mt lai vt liu nhi ct, ty theo kch c ht nhi ct, hng ch to ct thnh phn pha ng cng c th phi thay i cho ph hp 27

nh hng khi tng phn cc

20% H2O 30% H2O 40% H2O

Pha ng : MeOH

1 : p-Hydoxymethylbenzoate 2 : p-Hydoxyethylbenzoate 3 : p-Hydoxypropylbenzoate 4 : p-Hydoxybutylbenzoate

28

nh hng ca cc pha tnh

C8 C18 (ODS) Mnh sample Yu sample
29

Trung bnh sample C4

nh hng ca cc pha tnh

ODS C8 TMS

Analytical Conditions

Column : Shim-pack CLC-ODS Mobile phase : MeOH : H2O = 7 :3 Flow rate : 1.0 mL/min Temperature : 40 C Injection volume : 10 uL Detection : UV-254 nm Peaks 1. Methyl benzoate 2. Ethyl benzoate 3. n-Propyl benzoate 4. n- Butyl benzoate

30

So snh Normal phase v Reversed phase

Thng s phn cc ca ct phn cc ca dung mi Th t ra gii Normal Phase Cao Thp Reverse Phase Thp Cao

Cht km phn cc Cht phn cc ra ra trc trc T ng phn cc dung Ra gii nhanh Ra gii chm hn mi hn

So snh Normal phase v Reversed phase

Normal Phase
Tch tt cc ng phn lp th, v d nh Vitamin E etc Thi gian lu km n nh

Reversed Phase
lp li thi gian lu tt . Pha tnh khng ng u nhau.

32

Sc k cp ion pha o
Reversed Phase Ion-Pair Chromatography
Cht ghp cp ion

33

Cc cht ghp cp
Ghp cp vi cc Anion Tetra-n-butylammonium hydroxide (TBA) Ghp cp vi cc Cation Butanesulfonic acid sodium salt (C4) Pentanesulfonic acid sodium salt (C5) Hexanesulfonic acid sodium salt (C6) Heptanesulfonic acid sodium salt (C7) Octanesulfonic acid sodium salt (C8) Decanesulfonic acid sodium salt (C10)
34

Tch cc Carboxylic Acid bng ghp cp ion

Column: bonded C18 Mobile phase: H2O/MeOH 1:1 tetrabutyl ammonium hydroxide

35

Tch cc hp cht Amino bng cch ghp cp

Peaks
1. Nicotinic acid 2. Nicotinamide 3. Pantothenate 4. Pyridoxine 5. Riboflavin Phosphate 6. Thiamine 7. Caffeine 8. Folic acid 9. Biotin 10. Riboflavin

Analytical Conditions

Column : Shim-pack CLC-ODS Mobile phase : [A] : [B] = 9 : 1 [A] 100 mM phosphate buffer (pH=2.1) 0.8 mM sodium octane sulfonate [B] acetonitrile Flow rate : 1.5 mL/min Temperature : 40 C Injection volume : 10 uL

36

Nhng lu quan trng trong sc k ghp cp ion

Loi cht ghp cp Nng cht ghp cp pH ca pha ng
R-COOH R-NH2 + H+ RCOO- + H+ (pKa=4.5) R-NH3+ (pKa=6.0)
37

nh hng ca cht ghp cp

Hexane Sulfonate Pentane Sulfonate

Mobile Phase: H2O/MeOH 1:1,with 0.005M ion pairing reagent and 1% HOAc
1 Maleic Acid 2 Phenylephrine 3 Phenylpropanolamine 4 Naphazoline 5 Phenacetin 6 Pyrilamine

38

nh hng ca nng cht ghp cp

39

Ra ct khi s dng TBA

Nu s dng cht ghp cp l amine t cp hoc TBA, ct phn tch cn phi ra sau khi kt thc phn tch. Dung dch ra hiu qu nht thng l mui sodium perchlorate, bi v sodium perchlorate c i lc rt mnh i vi cc hp cht amine. Dung dch vi thnh phn nh sau thng c s dng ra ct : Dung dch 0.1% phosphoric acid c cha 100 mM sodium perchlorate : methanol = 1 : 1

40

Sc k ion : Ion Exchange

Lc ion R N+ R R

Sample + + + + + + + Sample + + + + + +
41

SO
3

Ion Exchange
S dng trong lnh vc sinh hc (phn tch protein, peptide, amino acid) Sc k Ion : phn tch cc hp cht ion Bao gm 2 loi ct:
Ct trao i Cation
Strong Cation Exchange Weak Cation Exchange (SCX) (R-SO3 )

(WCX) (R-COO ) (SAX) (R4N+) (WAX) (DEAE diethyl

42

Ct trao i Anion
Strong Anion Exchange Weak Anion Exchange aminoethyl)

Phn tch Protein s dng ct WCX

Analytical

Conditions

: Shim-pack WCX-1 Mobile phase : [A] 20 mM phosphate buffer (pH=6.0) [B] A+0.25M sodium sulfate [A] - [B] 30 min linear gradient Flow rate : 1.0 mL/min Temperature : ambient Detector : UV-280 nm Injection volume : 10 uL Peaks 1. albumin 2. myoglobin 3. -chymotrypsinogen A 4. liponuclease A 5. lisozyme 43

Column

Nhng im quan trng cn lu trong sc k ion

pH ca dung dch m Nng ca dung dch m Phng php ra gii
ng dng : Isocratic Gradient pH Gradient lc ion
44

Sc k gel SEC
Sc gel (sc k size phn t) (SEC) thng c bit di tn : GPC (Gel Permeation chromatography) : sc k thm thu gel, thng s dng trong lnh vc polymer. GFC (Gel Filtration Chromatography) : sc k tinh lc gel, thng s dng trong lnh vc sinh ha.
45

Nguyn l ca SEC
Khng s dng hiu ng tng tc. Tch da trn s khc nhau v thi gian di chuyn ca cht.

46

Th t ra gii

SEC Column

47

Mc ch ca GPC / GFC
GPC
S dng xc nh trng lng phn t ca polymer.

GFC
Tch cc protein

48

Mi tng quan gia trng lng phn t (MW) v thi gian lu RT

Molecular Weight (LogMW)

Gii hn loi tr Gii hn thm thu

Time

49

Lp ng chun
Tim tng dung dch chun polymer c phn t lng khc nhau bit c mi quan h gia trng lng phn t v thi gian lu .
No. 1 2 3 4 5 6 7 8 9 10 11 time(min) 22.0 22.6 23.4 25.0 27.4 31.0 33.8 38.4 39.8 42.8 46.8 mol. wet. 5500000 1800000 860000 400000 160000 50000 20000 4000 2000 600 80
50

Tim mu tht
tnh ton MW, cn phi c phn mm GPC

51

Tch Protein s dng ct GFC

Analytical

Conditions

: Asahipak GFA-50 Mobile phase : 0.1 M sodium phosphate 0.1 M NaCl (pH=7.0) Flow rate : 0.5 mL/min Temperature : ambient Detector : UV-280 nm Injection volume : 10 uL Peaks 1. glutamate dehydrogenase 2. lactate dehydrogenase 3. enolase 4. adenylate kinase 5. cytochrome C

Column

52

Sc k tch ng phn quang hc

M ir ro r

H R C*
NH2

H *C
HOOC NH2

*Chiral Center

COOH

(L) form

(R) form

Tnh cht vt l ca cc cht ny ging ht nhau, ngoi tr s quay cc.

53

Tch cc ng phn quang hc cch 1

Cho 2 ng phn to dn xut vi cht hu trin to cht lng hnh
Cht i hnh

(L) + (R) (R) + (R)

(L)-(R) (R)-(R)

Cht lng hnh

Cc sn phm to thnh c th tch bng Sc k pha thun hoc pha o

54

Tch cc ng phn quang hc cch 2

Tch trc tip s dng ct Chiral - Pha tnh c gn mt ng phn quang hc bit. (R) (R)
Tng tc mnh Tng tc yu

(L)

(R)

55

Cch la chn loi sc k

Loi sc k Reverse Phase Dung mi s dng H2O/Buffer, ACN, MeOH Ging RP nhng thm cht to cp ion Hexane, CH2Cl2 Cht phn tch Cc cht trung ha hoc khng ion ha, tan trong hn hp nc/dung mi hu c Cc cht dng ion hoc c th ion ha Hn hp cc cht khng tan trong hn hp dung mi hu c nc. Cc ion v c, protein, axit nucleic
56

Ion-Pair RP

Normal Phase

Ion Exchange

H2O/m

Cch la chn loi sc k

Lai sc k GPC/GFC Chiral Dung mi s dng H2O, THF, DMF, . Hn hp dung mi hu c nc Cht phn tch Cc cht c phn t lng ln Cc ng phn quang hc

57

Cc cch ra gii trong HPLC

Ra gii vi ch Isocratic
Thnh phn pha ng khng thay i trong sut qu trnh ra gii.

Ra gii vi ch Gradient
Pha ng vi thnh phn thay i trong qu trnh chy.
58

Cc cch ra gii trong HPLC

Isocratic
B concentration B concentration

Gradient

Time

Time
59

Isocratic
MeOH / H2O = 6 / 4

Thi gian phn tch di

Kh nng tch km
MeOH / H2O = 8 / 2
( ct C18 )
60

Gradient

95%

30%

% MeOH

61

u v nhc im ca ch Isocratic
u:
Thnh phn pha ng chnh xc. H thng phn tch n gin, r tin. ng nn n nh do thnh phn khng i nn s dng tt cho cc u d thnh phn nn nh hng nhiu n tn hiu nh dn (CDD), o ch s khc x (RI) Thnh phn pha ng c th trn trc bng tay hoc trn lin tc (nu c b gradient)
62

u v nhc im ca ch Isocratic
Nhc:
Kh nng tch km, c bit trong cc mu phc tp. Vic kho st thay i thnh phn pha ng cho ph hp thnh phn mu khng th thc hin nhanh. Thi gian phn tch di nu mun tch tt. Nu trn trc (khi khng c b gradient p sut thp) thnh phn pha ng c th thay i theo thi gian khi lu
63

u v nhc im ca ch Gradient
u:
Kh nng tch tt. Thnh phn pha ng kh chnh xc, c bit vi h gradient p sut cao. C th thay i rt nhiu thnh phn trong pha ng. chnh xc ca thnh phn theo thi gian gn nh khng i Thi gian phn tch kh ngn
64

u v nhc im ca ch Gradient
Nhc:
Thit b t tin hn, c bit l h gradient p sut cao. chnh xc ca thnh phn pha ng km hn h isocratic. Khi mt thnh phn c t l nh, thnh phn pha ng s km chnh xc v khng n nh.
65