Carnegie Mellon

The Hendrich Metalloprotein Group


How to prepare an EPR sample

Cleaning and calibration of X-band EPR tube
There are several sizes of EPR tubes; we use tubes with ID 3mm, OD 4 mm. Do not use tubes with OD 5mm since those will restrict helium flow. For quantitative sample analysis, the cross-sectional area of each sample tube needs to be determined. To calculate this area, fill an empty dry tube with known volume of water (e.g. 300uL) and measure the height of the solution in the tube. The volume (microliters) divided by the height (mm), is the cross-sectional tube area (mm2). Please include this calibration for each tube in the samples description. Small metal contaminations might result in additional resonances in the EPR spectrum. To avoid this, EPR tubes must be cleaned carefully. Clean the tubes with metal free hydrochloric acid and doubly distilled water. Make sure to rinse the tubes with plenty of 2D water. Dry the tubes in the oven. (You might want to check with NMR people around, they are likely to have a tube cleaning device, which will simplify cleaning).

Sample preparation and freezing
The sample height of solution in an X-band EPR tube should be a little greater than 25 mm (about 200 uL). The tube should not be filled more than 40 mm in height. When protein limited, it is possible to do measurements with smaller sample volumes (contact us for details). Transfer the sample into the EPR tube using either a syringe or a long glass pipette to prevent loss of the sample on the walls of the tube. Freezing of the sample in liquid nitrogen should be done very carefully. First, hold the very tip of the tube in liquid nitrogen (LN2) for 15 sec, then over 2 min, slowly immerse the rest of the sample into LN2. This will allow for sample expansion upwards during freezing, thus preventing the tube from cracking. After freezing is complete, check the tube to make sure that it’s not cracked.

Labeling the samples
If shipping multiple samples at a time, it’s important to label the tube properly. Do not use Scotch tape, it does not stay on the tubes when frozen. Ideally use hockey or medical type tape to make the labels. Wrap the tape tightly with no more than a 5 mm length sticking out sideways. Make sure you correlate the tube area calibration with the label. Also, it is good practice to use a grease pen to write an identifying number directly on the tube in case the label is lost.

Shipping frozen samples in EPR tubes
If you are shipping samples in a dry transport dewar, the dewar should have held LN2 overnight. Then prior to shipping, make sure that the liquid nitrogen is completely drained (shippers will often return due to residual LN2). Fill the empty space near the samples with paper towels to secure the tubes in the dewar to prevent breakage.

If shipping the samples on dry ice, do not put EPR tubes directly on dry ice, they will crack. Instead put the tubes inside rigid PVC tubing, secure the epr tube with Kimwipes within it, and tape both ends of the PVC tube before putting it on dry ice.

Do not send samples later than Wednesday on any given week. We do not want samples to be stacked somewhere over the weekend. Please email us the tracking number for the shipment as soon as you know it. Please email us as much detailed sample information as possible, including sample concentration, buffer/solvent, pH, preparation date etc… If you are missing some information or have questions, please call us!

Shipping Address:

If shipping samples that need to be kept cold, please write “store cold” on the package.

Prof. Michael Hendrich
Department of Chemistry
Carnegie Mellon University
4400 Fifth Ave.
Pittsburgh, PA 15213