## 4 Discussion  
### ADPH Extraction and Purification  
All the steps in the ADPH extraction and purification have been designed largely to prevent the breakdown of the compound before it can be isolated and carefully stored. The entire process needs to be done all in one day to ensure a minimal amount of hydrolysis occurs before the purification is complete. ADPH in any solution phase is not very stable and will break down readily which is why as many steps as possible are done on ice. All the centrifuge tubes, or samples of ADPH that are being processed must be kept on ice through the duration of the extraction and purification steps. It is stored as a solid at \(-80\)\({}^{\circ}\)C and is made into 1 mL stock solutions directly before use. When broken down, the once yellow solution will become very obviously pink. To prepare the purification elution buffer, triethylamine bicarbonate, a 1 L bottle of 750 mL H2O and 125 mL TEA is stirred with constant bubbling of CO2 (g) until the solutions combine thoroughly which typically takes about an hour. Unless there are extreme variations in the growing process, the extraction remains relatively consistent with little variability found in the purification. DEAE resin used must be cleaned and equilibrated between runs to maintain consistency which can be done according to the manufacturer's specifications. Oftentimes, purifications set on FPLC can be done without supervision however, it is crucial that once the fractions have been collected that they are immediately stored at \(-80\)\({}^{\circ}\)C until ready for lyophilization. When determining which fractions have ADPH by using mass spectrometry, there may be some complications with the instrument due to the sugars caramelizing in the electrospray chamber. To avoid this, run as few samples as possible; this is done by picking one or two fractions that represent the peak maxima for each species that elutes. In addition, it will help to flush the injection port with a 50:50 water:methanol mixture between samples to minimize clogs in the capillary. Thorough cleanings of the mass spectrometer sample chamber after using to assay for ADPH content should be done as well to avoid contamination of other samples. Successive lyophilization with pH 8 water has shown to decrease triethylammonium bicarbonate traces on the solid that can alter certain experimentation if introduced. It is also possible to determine the presence of ADPH in the samples using \({}^{31}\)P NMR or \({}^{1}\)H NMR [31; 32; 33]. The high throughput screening methods of ESI-MS make it far more suitable for ADPH extraction, however, without access to a mass spectrometer an NMR will be more than sufficient. ADPH can be stored as a solid at \(-80\)\({}^{\circ}\)C for upwards of a year with semi-regular checks to ensure small-molecule integrity has been maintained. It will last about three months as an aqueous stock at \(-80\)\({}^{\circ}\)C however, enduring freeze-thaw cycles will decrease its shelf life considerably with about 5-8 freeze-thaws before total product breakdown occurs.  
Figure 7: (**A**) Kdo\({}_{2}\)-Lipid A after lyophilization; (**B**) O-deacylated Kdo\({}_{2}\)-Lipid A after lyophilization.