CROMATOGRAFIA DE LICHIDE DE

NALT PERFORMAN (HPLC)

In alegerea metodei cromatografice trebuie avut in vedere urmatoarele:

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Schema de principiu

Agilent
Varian
Waters

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Aparatura HPLC

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Aparatura pompa HPLC

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Aparatura pompa HPLC

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Aparatura pompa HPLC

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Aparatura pompa HPLC

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Aparatura pompa HPLC

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Aparatura pompa HPLC

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Aparatura pompa HPLC

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Aparatura pompa HPLC

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Aparatura pompa HPLC

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Aparatura pompa HPLC

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Sistemul de injecie

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

Waters 717plus Autosampler

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Coloane cromatografice
coloana analitic pentru separare
- construit din oel
- dimensiuni: L 30-300 mm, diametrul 2,1-4,6 mm
- permite umpluturi cu dimensiunea particulelor
de 3-10 m
precoloana (coloana de gard)
- precede coloana analitic i evit contaminarea
acesteia
- conine aceeai umplutur ca i coloana analitic
- este mai scurt (cca 7,5 mm) i mai ieftin

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Faza staionar
un lichid legat covalent de particulele umpluturii
coloanei
- legarea covalent reduce deteriorarea prin
eluie a fazei mobile
- reacia dintre silicagel i derivai organici de
clorosilan, Si(CH3)2RCl

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Faza staionar
proprietile fazei staionare depinde de natura lui
R din grupa legat
1. R polar i faza mobil nepolar
(cromatografie n faz normal)
- ciano (-C2H4CN)
- diol (-C3H6OCH2CHOHCH2OH)
- amino (-C3H6NH2)
2. R nepolar i faza mobil polar
(cromatografie n faz inversa)
- lanuri hidrocarbonate octil (n-C8) sau
octadecil (n-C18)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Faza staionar
Schimb ionic
- Cationic
- Puternic acid sufonic
- Slab carboxil
- Anionic
- Puternic amin cuaternar
- Slab amin primar
Excludere
- Geluri
- Silicagel sau sticl cu mrime controlat

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Faza staionar
Silica Gel

Derivatized Silica Gel

Where R = C18H37
hydrocarbon chain
(octadecylsilyl deriv.
silica or C18)

bulk (SiO2)x

surface

relatively polar surface

normal phase

bulk (SiO2)x

surface

relatively nonpolar surface

reversed phase

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Faza mobil
eluia analiilor este dat de polaritatea
fazei staionare i a celei mobile
cromatografia n faz normal
- analiii nepolari elueaz primii
- creterea timpilor de retenie prin scderea
polaritii fazei mobile
cromatografia n faz invers
- analiii polari elueaz primii
- creterea timpilor de retenie prin creterea
polaritii fazei mobile

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Alegerea fazei mobile
polaritatea solvenilor dat de indicele
de polaritate (P)
- valori mari corespund solvenilor mai polari
- prin amestecarea a 2 sau mai muli solveni se
obine un amestec cu polaritate intermediar:
PAB = APA + BPB
unde PA i PB sunt indicii de polaritate iar
A i B sunt fraciile volumelor solvenilor A i B

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Proprietile fazelor mobile

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Normal vs. Reversed Phase Chromatography

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Tipuri de eluie
a) eluia izocratic compoziia fazei mobile este constant n
decursul analizei
b) eluia n gradient compoziia fazei mobile se modific n
timp:
- pentru cromatografia n faz invers, compoziia iniial este
polar
- polaritatea descrete n timpul separrii

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Selectia coloanelor
Chromatographic Column Parameters
The six chromatographic column parameters are determined experimentally.
2.1.1 Retention factor for pentylbenzene, kPB
A measurement of the surface area and surface coverage (ligand density).
Chromatographic Conditions: MeOH-H 2O (8:2, v/v), 1.0mL/min, 40C, 5L injection of pentylbenzene
(0.6g/mL). Injections and flowrates have been scaled for 4.6 mm i.d. columns.
The first disturbance of the base-line when injecting methanol has been used as a dead time marker.
2.1.2 Hydrophobicity or hydrophobic selectivity, aCH 2
The retention factor ratio between pentylbenzene and butylbenzene, aCH 2 = kPB/kBB, is a measure of the surface
coverage of the stationary phase as the selectivity between alkylbenzenes differentiated by a single methyl group
depends on ligand density.
Chromatographic Conditions: individual injections of pentylbenzene (0.6g/mL) and butylbenzene (0.3g/mL).
2.1.3 Shape selectivity, aT/O
The retention factor ratio between triphenylene and o-terphenyl, aT/O = kT/kO, is a measure of the shape
selectivity, which depends on ligand spacing and the shape/functionality of the silylating reagent.
Chromatographic Conditions: All conditions as for aCH 2 determinations, except 5L injection of o-terphenyl and
triphenylene both at 0.05mg/mL.
2.1.4 Hydrogen bonding capacity, aC/P
The retention factor ratio between caffeine and phenol, aC/P = kC/kP, is a measure of the
number of available silanol groups and the degree of endcapping.
Chromatographic Conditions: MeOH-H 2O (3:7, v/v), 1.0ml/min, 40C, individual 5L of phenol

CROMATOGRAFIA DE LICHIDE DE
NALT PERFORMAN (HPLC)
Selectia coloanelor
Chromatographic Column Parameters
2.1.5 Total ion-exchange capacity, aB/P pH 7.6
The retention factor ratio between benzylamine and phenol, aB/P pH 7.6 = kB/kP, is a measure of
the total silanol activity.
Chromatographic Conditions: 20mM KH2PO4, pH 7.6, in MeOH-H2O (3:7, v/v), 1.0mL/min, 40C,
individual 5L injections of phenol and benzylamine HCl both at 0.5mg/mL.
2.1.6 Acidic ion-exchange capacity, aB/P pH 2.7
The retention factor ratio between benzylamine and phenol, aB/P pH 2.7 = kB/kP, is a measure of
the acidic activity of the silanol groups.
Chromatographic Conditions: All conditions as for total ion-exchanged determinations above,
except using a pH 2.7 KH2PO4 buffer.
2.2 Calculation Methods
The Chromatographic Column Selector program contains the above six parameters for 135
chromatographic columns.