Freeze drying sample holders

Figure 8-5. Freeze-drying sample holder. (Courtesy - Labconco Co., Kansas City, MO)...

X-ray photoelectron (ESCA) spectra of carbenium ions have also been obtained in frozen superacid solutions, generally in SbF5-S02 solution or as isolated salts. Sulfur dioxide was subsequently removed by the usual freeze-thaw procedure. A thin layer of the viscous SbF5 solution was deposited on the precooled sample holder, in a dry nitrogen... 

The sensitivity quoted above is for a dry sample and this is greatly reduced by the presence of water. This raises difficulties in the study of samples such as tissue slices, blood, or cell cultures in in vivo conditions. Specially shaped sample holders have to be used and the sensitivity is reduced by a factor of about 100 (allowing for the smaller volume of sample which can be introduced into the spectrometer). A way round this difficulty is to freeze the sample (which restores the full sensitivity), and then to record the spectrum at a low temperature, usually that of liquid nitrogen (77°K). 

Since the thimble and the freeze-dried mass could hinder mass transfer, another set of experiments were conducted at 24 and 31 MPa In the cups of an aluminum sample holder with six cups (0.5 cm x 0.5 cm). It was reasoned that If the thimble was eliminated from the extraction, and If only the lipids were exposed to the SC CO2, there would be negligible mass transfer resistance to extraction. Solvent extracted lipids were placed In the cups, and extracted with SC CO2. 

Effect of Phospholipase C on Hold yield. Phospholipase C from Clostridium oerfrlnaens (Sigma Chemical Company, St. Louis, MO) was used to cleave the phosphate group of Skeletonema lipids, and to change the polarity of phospholipids to Increase the SC CO2 extraction yield. Six grams of freeze dried Skeletonema were extracted with solvents (21). The lipids obtained were split Into three fractions. The first fraction was used to determine the fatty acid profile of the lipid to serve as a basis. The second fraction was deposited onto the sample holder. The lipids were extracted with SC CO, as control at 31.0 MPa. The third fraction was treated with phospholipase C In Trizma buffer (Sigma Chemical Company, Saint Louis, MO) and Incubated at pH 3.7 at room temperature for 3 hours (22). The treated lipids were deposited onto the sample holder and extracted with SC CO2 at 31 MPa. The extracted lipids were compared with the profile lipids and with the lipids from the control experiment. In both SC extraction cases, the SC CO2 extracted lipids and the lipids remaining on the sample holder were compared by GC analysis. 

Table IV. Total Lipid Mass Balance experiments in sample holder for freeze dried Skeletonema costatum...

Aqueous raffinose and trehalose solutions (400 mmol dm ) were dropped separately on x-ray sample holders. These samples were precooled at —85° for 30 min and then freeze-dried for 20 h in the condition of below 1.33 Pa. After freeze-drying, the samples were determined by a microbalance and an x-ray diffractometer. Then the samples were stored in desiccators at 35° containing saturated salt solutions with different relative humidities (RHs). After a certain interval storage at each of two humid conditions, the samples were determined by a microbalance and an x-ray diffractometer. RHs of saturated Mg(N03)2 and NaCl solutions at 35° are 52 and 76%, respectively. 

The technique of filling the sample holder is to fill it 1/3 to 1/2 full of liquid. Place the lower end into the dry ice-acetone or isopropanol container, and tilt the holder to about a 60 angle or as flat as possible without spilling the liquid. Slowly turn the holder so a film of solid sample freezes around the inside of the sample holder. Freeze the sample for a few minutes longer, connect the top section and then connect it to the condenser before it begins to melt. This rotation technique increases the surface area, and the water in this thin film, about 1 cm thick at most, can be sublimed rapidly. 

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