INVERSE 2D-CORRELATION

The Inverse HX-correlation experiments use pulse sequences which are set up to pulse both protons and an X-nucleus, e.g., carbons. The proton spectrum is observed. Actually, the pulse sequences normally suppress the protons not bound to the X-nucleus. For proton-carbon experiments, the protons bound to ¹²C are suppressed, thus leading to a carbon satellite spectrum.
The experiments can be designed with decoupling of the X-nucleus, solvent suppression, long-range correlation etc.
Inverse proton-phosphorus experiments have been successfully used as well. An Inverse HX-correlation experiment on a 5 mg sample of rotenone (MW ť 384) takes about 50 minutes on the AMX-300!

Calibrating The 90 Degree Decoupler Pulse

To set up the experiment for the first time, one needs to calibrate the 90 º pulse for the X-nucleus. We can use a pulse program to observe the effect of the X pulse on the X satellites in the proton spectrum.

The pulse program decp90 is shown schematically below:

To calibrate the 90 º decoupler pulse for ¹³C, we can use, e.g., CHCl₃. The 1/2J delay D6 is set to about 2.3 ms. P1 is the 90 º observe pulse at the power selected by DP, P3 is the decoupling pulse.
By varying P3, we can note the change of intensity of the satellite doublet in the proton spectrum. For P3 = 0, the satellites are of normal intensity. As P3 is increased, the intensity decreases. At P3 = 90 º , the doublet is nulled. For P3 > 90 º, the doublet inverts and the intensity increases again.

The proton parameters are set up as usual. For pulsing carbon, it is best to set SFO2 to be on-resonance (near 77 ppm for CHCl₃) to reduce errors due to off-resonance effects. This is particularly important if low power pulses need to be calibrated!

An easy way to set up approximate parameters is to use the edsp feature. Click on [Param] > [Spectrometer] > [edsp] . Select Observe Nucleus 1H and Decouple Nucleus ¹³C. The exact values for SFO1 and SFO2 will need to be corrected, use values obtained from previous 1D spectra.

For low power X-decoupling, the same basic experiment is used to determine the low power 90 º pulse, except that the decoupler is set to low power in the decp90lo experiment. The power for the carbon pulses is set by selecting the value for DL0. A reasonable DL0 level to start is in the range of 25 to 30. The decoupling range in Hz is about 5 times the B1 field strength. On a 400 MHz QNP- probe a 90 º of 60 µ sec has been measured at DL0 of 28.
The effective decoupling range is about 20 kHz at 60 µ sec (200 ppm at 100.61 MHz). Shorter pulses are not necessary; due to the higher power the sample temperature will increase and the decoupling band width is wider than needed. Conversely, too long a pulse will lead to marginal decoupling. For most applications a width of 40 µ sec will be short enough for an AMX-600 while 80 µ sec will work well on an AMX-300 etc.

Hardware

Note: Make sure that the Proton Band Pass Filter (e.g. 300 MHz Band Pass on the AMX-300) is installed between the preamp 1H connector and the probes' 1H connetcor! Otherwise you will get garbage if X-decoupling is used!
he filter is attached at the probe or the preamp housing connector.

Only one minor hardware change is required. The 2H-Stop filter must be connected between the probe (X-nucleus port) and the regular filters at the pre-amp input. This is to prevent X-decoupling noise from interfering with the lock signal.

In principle, the 2H-Stop filter can be left in place except for measuring Deuterium or nuclei within about +/- 5 MHz of Deuterium!

Parameter files:

invcar.400 for VBAMX-400, invcar.300 for AMX-300.

Parameter Example for AMX-400

A typical experiment (at 400 MHz) can be set up with a 2K acquisitions size in F2 (protons) and 256 or 512 increments. This leads to a digital resolution of about 2 Hz for protons and 20 or 10 Hz for carbon. Processing is done with a squared cosine bell (QS, SSB = 2).
The phase parameters for the F2 (proton) dimension can be obtained from the normal proton spectrum provided the same receiver gain is used.

Note: If the proton SW is changed, the loop count L2 must be re-calculated! Use xau GARPLOOP or the equation given in the pulse program.

The Bruker calcphinv xau program can be used to calculate the 1st. order phase correction in F1.

F2 - Acquisition Parameters

PULPROG	invbdgtp
TD		2048
NS	8
DS	4
SW	11.058 ppm
SWH	4424.78 Hz
HZpPT	2.160537 Hz
AQ	113.0  µsec
SOLVENT	7.25
NUCLEUS	1H
BF1	 400.13 MHz
O1	6300.00 Hz
DECNUC	13C
BF2	100.62 MHz
O2	1600.00 Hz
TE	297 K
AQ_mod	qseq
RG	512
DR	16
HL1	0 dB
DECSTAT 	DO

F1 Acquisition parameters

ND0	4
SW 	207.044 ppm
SFO1	100.6227320 MHz
HZpPT	20.345053 Hz
TD	256

F2 - Processing parameters

SI	2048
SF	400.1344137 MHz
WDW	QSINE
SSB	2
LB	0.00Hz
GB	0
PC	4.00
OFFSET	10.243 ppm

F1 - Processing parameters

MC2	TPPI
SI	1024
SF	100.6150790 MHz
WDW	QSINE
SSB	2
LB	0.00 Hz
GB	0
OFFSET	179.592 ppm


Other Parameters Used On AMX-400: 11/18/89

P1 = 9.5        P2 = 19    P3 = 4.5    P4 = 9
P9 = 60 @DL0 = 28
D1 = 1.5 sec    D2 =3.44 msec D7 = 0.45 sec

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Questions, comments to: Rudi Nunlist rnunlist@bloch.cchem.berkeley.edu

Last Update: 10/18/95. RN