INSTRUMENTELE ANALYSE 2:
SCHEIDINGSTECHNIEKEN
Lector. Opsteyn Ine
2023 - 2024
,INHOUD
1. Chromatografische technieken : inleiding .......................................................................................................... 3
1.1 Indeling van de chromatografische technieken ............................................................................................ 3
2. Gaschromatografie (GC) ...................................................................................................................................... 4
2.1 Verloop van een GC scheiding....................................................................................................................... 4
2.2 GC-scheidingsprincipe ................................................................................................................................... 4
2.3 Instrumentatie .............................................................................................................................................. 5
2.3.1 Dragergas ............................................................................................................................................... 5
2.3.2 Injectie .................................................................................................................................................... 5
2.3.3 Capillaire kolom...................................................................................................................................... 6
2.3.4 Oven ....................................................................................................................................................... 6
2.3.5 Detector – FID ........................................................................................................................................ 8
2.3.6 Registratie detectorsignaal .................................................................................................................... 9
Oefeningen ........................................................................................................................................................ 11
3. Vloeistof chromatografie (LC) ........................................................................................................................... 12
3.1 Algemeen .................................................................................................................................................... 12
3.2 Scheidingsprincipe ...................................................................................................................................... 12
3.2.1 Mobiele fase ......................................................................................................................................... 12
3.2.2 Stationaire fase .................................................................................................................................... 13
3.2.3 Injectie .................................................................................................................................................. 15
3.2.4 Pomp .................................................................................................................................................... 15
3.2.5 Detector ............................................................................................................................................... 16
4. Kwantifacatiemethoden .................................................................................................................................... 18
4.1 Interne standaard methode ........................................................................................................................ 18
4.1.1 Eenpunts kalibratie .............................................................................................................................. 18
4.1.2 Ijklijnmethode ...................................................................................................................................... 19
4.2 Standaardadditiemethode .......................................................................................................................... 19
4.2.1 eenpunts kalibratie .............................................................................................................................. 19
4.2.2 ijklijnmethode ...................................................................................................................................... 19
1
, 5. Kwaliteit van scheidingen.................................................................................................................................. 21
5.1 het chromatogram ...................................................................................................................................... 21
5.2 Kwaliteit van scheidingen ............................................................................................................................ 21
5.2.1 Schotelgetal .......................................................................................................................................... 21
5.2.2 Resolutie............................................................................................................................................... 22
5.2.3 Assymetriefactor T (tailing factor) ....................................................................................................... 24
Oefeningen ........................................................................................................................................................ 25
6. hoge resolutie Elektroforese ............................................................................................................................. 27
6.1 Elektroforese als scheidingstechniek .......................................................................................................... 27
6.2 Gelelektroforese.......................................................................................................................................... 27
6.2.1 Scheidingsfactoren ............................................................................................................................... 27
6.2.2 Zuurtegraad van de oplossing .............................................................................................................. 27
6.2.3 Diffusie ................................................................................................................................................. 28
6.3 (Iso)-elektrofocusing (IEF) ........................................................................................................................... 28
6.3.1 Basisprincipe ........................................................................................................................................ 28
6.3.2 pH-gradiënt .......................................................................................................................................... 29
6.3.3 Apparatuur ........................................................................................................................................... 29
6.3.4 Dunlaag elektrofocusing ...................................................................................................................... 29
6.4 Gelelektroforese VS Elektrofocusering ....................................................................................................... 30
2
SCHEIDINGSTECHNIEKEN
Lector. Opsteyn Ine
2023 - 2024
,INHOUD
1. Chromatografische technieken : inleiding .......................................................................................................... 3
1.1 Indeling van de chromatografische technieken ............................................................................................ 3
2. Gaschromatografie (GC) ...................................................................................................................................... 4
2.1 Verloop van een GC scheiding....................................................................................................................... 4
2.2 GC-scheidingsprincipe ................................................................................................................................... 4
2.3 Instrumentatie .............................................................................................................................................. 5
2.3.1 Dragergas ............................................................................................................................................... 5
2.3.2 Injectie .................................................................................................................................................... 5
2.3.3 Capillaire kolom...................................................................................................................................... 6
2.3.4 Oven ....................................................................................................................................................... 6
2.3.5 Detector – FID ........................................................................................................................................ 8
2.3.6 Registratie detectorsignaal .................................................................................................................... 9
Oefeningen ........................................................................................................................................................ 11
3. Vloeistof chromatografie (LC) ........................................................................................................................... 12
3.1 Algemeen .................................................................................................................................................... 12
3.2 Scheidingsprincipe ...................................................................................................................................... 12
3.2.1 Mobiele fase ......................................................................................................................................... 12
3.2.2 Stationaire fase .................................................................................................................................... 13
3.2.3 Injectie .................................................................................................................................................. 15
3.2.4 Pomp .................................................................................................................................................... 15
3.2.5 Detector ............................................................................................................................................... 16
4. Kwantifacatiemethoden .................................................................................................................................... 18
4.1 Interne standaard methode ........................................................................................................................ 18
4.1.1 Eenpunts kalibratie .............................................................................................................................. 18
4.1.2 Ijklijnmethode ...................................................................................................................................... 19
4.2 Standaardadditiemethode .......................................................................................................................... 19
4.2.1 eenpunts kalibratie .............................................................................................................................. 19
4.2.2 ijklijnmethode ...................................................................................................................................... 19
1
, 5. Kwaliteit van scheidingen.................................................................................................................................. 21
5.1 het chromatogram ...................................................................................................................................... 21
5.2 Kwaliteit van scheidingen ............................................................................................................................ 21
5.2.1 Schotelgetal .......................................................................................................................................... 21
5.2.2 Resolutie............................................................................................................................................... 22
5.2.3 Assymetriefactor T (tailing factor) ....................................................................................................... 24
Oefeningen ........................................................................................................................................................ 25
6. hoge resolutie Elektroforese ............................................................................................................................. 27
6.1 Elektroforese als scheidingstechniek .......................................................................................................... 27
6.2 Gelelektroforese.......................................................................................................................................... 27
6.2.1 Scheidingsfactoren ............................................................................................................................... 27
6.2.2 Zuurtegraad van de oplossing .............................................................................................................. 27
6.2.3 Diffusie ................................................................................................................................................. 28
6.3 (Iso)-elektrofocusing (IEF) ........................................................................................................................... 28
6.3.1 Basisprincipe ........................................................................................................................................ 28
6.3.2 pH-gradiënt .......................................................................................................................................... 29
6.3.3 Apparatuur ........................................................................................................................................... 29
6.3.4 Dunlaag elektrofocusing ...................................................................................................................... 29
6.4 Gelelektroforese VS Elektrofocusering ....................................................................................................... 30
2
