The earliest HPC work used the oil-coating method. However, some crystals are damaged by oil coating, and the capillary shielding method was developed as an alternative. Damage can also occur during the manipulations required by this second method, so we will try both methods when working with a new crystal type. Typically 4-6 crystals will be processed with each method, in order to establish the preferred method for the crystal type.
Procedure:
For oil-coating
- Under a microscope, completely cover a drop of mother liquor containing crystals with oil (usually with NVH oil).
- Gently drag a crystal are gently dragged from the droplet into the oil.
- Pick up the crystal in a cryoloop (Hampton Research 18mm Cryoloop), so the crystal (and a little bit of mother liquor around the crystal) is coated with oil. Slightly touch the cover slide to remove excessive oil if there is too much oil around the crystal (which will increase the diffraction background and prevent visualizing crystal when centering it).
- Without magnification, insert the Cryoloop with mounted crystal into a special stainless steel pin-holder.
- Skip to step 10.
For capillary shielding:
- Cut polyimide capillary to fragments of 2-3cm long. One end of the capillary is sealed with epoxy, then filled with ~20ul mother liquor with a syringe (be careful not to trap air bubbles between the epoxy and the mother liquor).
Insert a Cryoloop (18mm, Hampton Research) into a stainless steel pin-holder. Apply a little bit of epoxy between the Cryoloop and the inner surface of the holder to prevent the Cryoloop from falling out of the holder during pressurization.
- Pick up a crystal from the mother liquor drop with the Cryoloop-holder assembled in step 7. No cryoprotectant is need.
- Insert the Cryoloop-holder (with crystal) into the capillary prepared in step 6. A plastic plate with a guiding groove will help this insertion go accurately and efficiently.
- Fit one end of a length of high pressure tubing (High Pressure Equipment Company, Erie, PA, USA) with a removable endcap; torque to 20 lbs pressure. Orient the tubing with the endcap at the bottom and slide the Cryoloop-holder assembly into the top of the tube with the mounted crystal pointing up. An external rare-earth magnet is positioned to hold the stainless steel holder, and thus the Cryoloop with the mounted crystal, near the top.
- Up to three high pressure tubes can be mounted on the high pressure cryocooling apparatus at a time, with the endcap extending down ~5 cm into a bath of liquid nitrogen.
- Connect the high pressure tubes to the gas compressor, using 20 lbs torque pressure to secure connections.
- Pressurize system with helium to desired pressure, usually 200-400 MPa. Allow system to equilibrate 10-30 minutes. This ensures crystals are properly pressurized uniformly.
- Release the magnet, letting the Cryoloop (together with the crystal, holder and capillary) fall to the bottom of the high pressure tube, which is immersed in liquid nitrogen. You should notice a small positive change in pressure.
- Allow system to equilibrate ~2 minutes. This ensures your samples are properly cooled uniformly.
- Release pressure from the system.
- Disconnect tubes at top and bottom, keeping Cryoloops with crystals under liquid nitrogen. (CAUTION – If samples are not kept under liquid nitrogen, the slight change in temperature will release the pressure within the crystals, thus the trapped helium gas, and destroy the crystal.)
- For those crystals pressurized by the oil-coating method, under liquid nitrogen, pull the Cryoloop out from the holder using long forceps and mount it to a standard base. For those crystals pressurized by the capillary shielding method, under liquid nitrogen, remove the shielding capillary with long forceps, then mount the Cryoloop-holder to a MX HP base (MiTeGen).
- Handle and store pressurized crystals like any other cryocooled crystals.
- Mount crystal onto a goniometer at the x-ray beamline under coldstream as quickly as possible.
Collect diffraction images and analyze as a normal sample.