5 MATERIAL UND METHODEN
5.3 Methoden
5.3.5 Kernresonanzspektroskopie (NMR)
Direktinjektion
Der Probeneinlass über Direktinjektion (Spritzenpumpe) erfolgte im Allgemeinen mit den nachfolgenden Einstellungen (lowflow-Methode):
Kapillarspannung (Capillary voltage): ± 3800 V (polaritätsabh.) End plate offset: ± 500 V (polaritätsabh.) Vernebelungsgas (Nebulizer gas): 5 psi
Trockengas (Dry gas): 5 L/min Trockengastemperatur (Dry heat): 300 °C
Eventuelle Abweichungen von diesen Parametern werden im Ergebnisteil des betreffenden Kapitels genannt.
Die Parameter „Target mass“ (TM) und „Compound stability“ (CS) wurden abhängig von der untersuchten Lipidklasse sowie der Stabilität des Mutterions variiert. Dabei dient der Parameter „Target mass“ dazu, die Empfindlichkeit im Bereich des gewählten m/z Wertes zu verbessern. Der Parameter „Compound stability“ stellt ein Maß für die Stärke der Fragmentierung dar, die bereits im Transferbereich zwischen Ionenquelle und Ionenfalle durch Kollisionsinduzierten Dissoziation (Skimmer-CID) erfolgt. Die CS-Werte können zwischen 10 und 1000 liegen, wobei in dieser Arbeit zumeist CS-Werte zwischen 30-100 gewählt wurden.
Durch Tandem-MS-Experimente (analog MS/MS-oder MSn-Experimente) werden Ionen eines bestimmten m/z-Werts in der Ionenfalle isoliert, um diese anschließend zu fragmentieren. Dies gelingt durch eine zusätzliche Wechselspannung auf den Endkappen der Ionenfalle, wodurch die isolierten Ionen angeregt und Stöße mit Inertgas (z. B. Helium) induziert werden. Hierbei stellt der Parameter „Amplitude“ (A) ein Maß für die Stärke der Fragmentierung dar. Aus den erhaltenen Fragmentionen kann wiederum ein Fragment ausgewählt werden, das isoliert und fragmentiert wird. Da jeder dieser Schritte mit einem Intensitätsverlust einhergeht, ist die Anzahl an möglichen Fragmentierungs-schritten begrenzt.
Kopplung mit HPLC
Bei Anwendung von HPLC-MS-Kopplung sind die massenspektrometrischen Parameter bei der jeweiligen HPLC-Methode genannt.
Tabelle 33: Aufnahmebedingungen für die 1D-NMR-Experimente.
Parameter Abk. 1H (Übersicht) 1H (Quantifizierung) Anzahl Datenpunkte TD 32768-65536 32768 Spektrenbreite [ppm] SW 8.976 8.976 Mitte des Spektrums [ppm] O1p 3.500 3.500
Anzahl Dummy Scans DS 8 8
Anzahl Scans NS 64-256 64
Wartezeit [s] D1 3 22
Pulslänge [μs] P1 9.7 8.5
Pulsleistung [dB] PL1 4.0 2.0 Akquisitionszeit [s] AQ 6.081 3.04 Empfängerleistung RG 128-256 256-512
Tabelle 34: Aufnahmebedingungen für die 2D-NMR-Experimente.
Parameter Abk. HSQC HSQC-
TOCSY HMBC Anzahl Datenpunkte in F2 TD (F2) 4096 4096 4096 Anzahl Datenpunkte in F1 TD (F1) 1024 1024 1024 Spektrenbreite in F2 [ppm] SW (F2) 8.01 8.01 8.01 Spektrenbreite in F1 [ppm] SW (F1) 150.0 200.0 200.0 Mitte des Spektrums in F2 [ppm] O1p 4.00 4.00 4.00 Mitte des Spektrums in F1 [ppm] O2p 75.0 100.0 100.0 Anzahl Dummy Scans DS 32 32 32
Anzahl Scans NS 8 16 16
Wartezeit [s] D1 3.0 2.0 2.0
Pulslänge [μs] P1 9.4-9.7 9.7 9.7
Pulsleistung [dB] PL1 4.0 4.0 4.0
Mischzeit [ms] D9 - 20 60
Empfängerleistung RG 16-32k 32k 32k
Die Quantifizierung erfolgte mittels externem NMR-Standard Trimethylsilyl-propionat-Natriumsalz (TSP), auf den auch kalibriert wurde (0,00 ppm). Die Konzentrationsbestimmung der Standardkapillaren wurde vierfach mit den Substanzen Vanillin, N-Hydroxysuccinimid und Cholesterol durchgeführt.
Über die T1-Zeit-Bestimmung eines Standardgemisches mittels eines Inversion-Recovery-Experiments ließ sich die Wartezeit (ca. 5xT1) für das 1 H-Quantifi-zierungsexperiment berechnen. So ist sichergestellt, dass die Kerne vor dem nächsten Scan vollständig ausrelaxiert sind.
Zur Datenverarbeitung wurden die Programme Topspin 1.3 (Bruker) und MestReC 4.9.9.9 bzw. MestReNova (Mestrelab Research) verwendet.
Die aufgenommenen FIDs (free induction decay) wurden mittels einer linear prediction auf 64k aufgefüllt und mit einer Exponentialfunktion apodisiert. Die Phasenkorrektur erfolgte manuell, während für die Basislinienkorrektur der Witthaker Smoother verwendet wurde.
Die Zuordnung der Signale in den NMR-Spektren wurde anhand verschiedener 2D NMR-Experimente und durch den Vergleich mit aufgenommenen Referenzspektren von Standardsubstanzen (siehe Anhang II Charakterisierung von Standardsubstanzen) sowie durch Abgleich mit Literaturdaten vorgenommen (Sparling 89, Willker 98, Willmann 07).
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