Information on the sample requirements for the various instruments in the laboratory.
The following table details the volume and sample tube requirements for the various resonators and cavities available in the laboratory. Please note that whenever ordering EPR tubes, clear fused quartz is required. Glass or borosilicate tubes absorb microwaves and spoil the Q of the resonator, making tuning difficult or impossible.
| Resonator | Tube | Vendor | Minimum Volume |
Recommended Volume |
Notes |
| X-band CW Cavities |
707-SQ-250M | Wilmad | 175 μL | 200 μL | Volumes are for frozen samples, capillaries or a flat cell are required for room temperature aqueous samples (see below). |
| X-band Pulse ENDOR | 706-PQ-9.50 | Wilmad | 80 μL | 100 μL | Can be used for all pulse experiments including ENDOR. |
|
X-band |
705-PQ-6.25 | Wilmad | 20 μL | 25 μL | Can be used for all pulse experiments with the exception of ENDOR. |
| X-band 2 mm Split Ring |
1.1 mm ID 1.6 mm OD 100 mm Length |
Vitrocom | 6 μL | 10 μL | Can be used for all pulse experiments with the exception of ENDOR. Ideal for sample limited X-band experiments. Tubes need to be custom ordered and take several weeks to arrive. |
| Q-band Pulse - DEER |
705-PQ-6.25 | Wilmad | 20 μL | 100 μL | Can be used for pulse experiments excluding ENDOR. |
| Q-band ENDOR |
1.1 mm ID 1.6 mm OD 100 mm Length |
Vitrocom | 6 μL | 10 μL | Can be used for all Q-band pulse experiments including ENDOR. Ideal for sample limited Q-band experiments. Tubes need to be custom ordered and take several weeks to arrive. |
| X-band Power Saturation |
TPX Capillaries | Bruker | 5 μL | 20 μL | Very small gas permeable capillary tubes for power saturation studies. |
X-Band Sample Requirements
The target concentration to obtain a decent signal to noise ratio is usually 200 μM. Higher concentrations can be beneficial, however, concentrations that are too high can potentially lead to artificial line broadening and enhanced relaxation from spin-spin interactions. This can make pulse experiments very difficult. This is not as important for typical protein samples as the surrounding protein usually acts as a buffer isolating the individual spins.
The last important aspect to consider is the solvent. Organic non polar solvents can typically be used with standard sized X-Band tubes at all temperatures. Unfortunately, samples in aqueous solvents present some difficulties at temperatures above 0° C. The large electric dipole of water interacts strongly with the E field of the standing microwave formed within the resonator. EPR microwave resonators are designed to limit the density of this field at the sample, but it is not possible to completely isolate the sample. The interaction between the two adversely affects the performance of the instrument and the ability to tune the resonator. Therefore, aqueous samples require the use of a thin sample holder called a flat cell or a Q-Band tube if the signal is strong enough. Flat cells are available and various types can be purchased from Wilmad, depending on the individual sample requirements.
Q-Band Sample Requirements
As with X-Band samples, a target concentration of 200 μM is typically desired, however, Q-Band EPR is inherently more sensitive than X-Band, so more dilute concentrations can be used depending on the experiment.
For aqueous samples, very narrow quartz capillary tubes can be used with great success. Quartz capillary tubes can also be ordered from Vitrocom. A 0.4 mm OD and 0.3 mm ID quartz tube (CV3040Q) works well (make sure to order quartz and not fused silica tubes). The sample can be loaded by placing the tube in the sample reservoir. Capillary action will automatically load the tube to a proper height. The capillary can then be placed in a standard Q-Band tube.