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Samenvatting - GC & HPLC
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Gaschromatografie & HPLCSamenvatting
,Les 1
Retentietijd
Retentietijd = tijd die nodig is om een opgeloste stof te elueren tr = tm + t’r
Tr = (bruto) retentietijd
Tm = tijd van een onvertraagd component
= tijd van verblijf in de mobiele fase componenten die worden vertraag
T’r = netto retentietijd = tijd van verblijf in de stationaire fase
Retentiefactor voorheen capaciteitsfactor
Resolutie (scheiding)
Resolutie wordt bepaald door:
o Afstand tussen de pieken (tr)
o Breedte vd pieken (w)
Goede scheiding als:
o Δtr groot is
o W klein is
Voor kwantitatieve bepaling: Rs > 1,5
Rekenen met schotels, formules toepassen
( ) ( )
2 2
tr tr
o Theoretical plates: N=16 =5,545
wh wh
Lc
o Plate height: HETP=H=
N
o HETP: Theoretische schotelhoogte: "Height of a Theoretical Plate".
Schotels bij LC column
1000∗l
o Voor totaal poreuze kolom geldt theoretisch: N=
2∗dp
1000∗l
o In de praktijk: N=
2,5∗dp
o l lengte kolom in mm
o dp deeltjes grootte in micrometer
o
, Parameter Gegevens chromatogram Formule
Capaciteitsfactor k tr en tm
t r ' t r−tm
k= =
tm tm
Selectiviteit α tr en tm
t'r2 k2
α= =
t'r1 k1
[ ]
Schotelgetal N tr en σ 2
FWHM = 2.35σ tr
FWBP = 4σ N=16∗
FWBP
[ ]
2
tr
N=5,545∗
FWHM
Resolutie Rs tr en σ
∆tr (tr 2−tr 1)
Rs= = ¿¿
W
( tr 2−tr 1)
Rs=
0,85∗(FWHM 1+ FWHM 2)
Rekenen aan het chromatogram
Retentiemechanisme
o Adsorptie GSC
o normal phase
o Verdeling GLC
o reversed phase
o Elektrostatisch interactie
o Ion – exchange
o Moleculaire zeefwerking
o Biologische affiniteit
pKa / log p
o pH < pKa zuur neutral, base positief geladen
o log p < 1 polair
o 1< log p < 3 matig polair
o Log p > 3 apolair
Type chromatografie
Reversed phase
- Stationaire fase = apolair
- Mobiele fase = polair
- Veel gebruikte basis-eluens: water, eventueel met bufferzouten en/of
andere hulstoffen
- Veel gebruikte modifiers: methanol, acetonitril
Normal phase
o Stationaire fase = polair
o Mobiele fase = apolair
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