41 Precipitation
For the scope of this investigation, protein precipitation by acid and by water miscible organic solvents are explored, both of which rely on different chemical principles. Organic solvents cause precipitation of proteins by significantly lowering the dielectric constant of the whole blood solution, which causes the electrostatic interactions between proteins to increase. The solvent also removes any of the surrounding water shell that effectively minimises the hydrophobic interaction between proteins, thus causing electrostatic interactions to become paramount which leads to protein aggregation [5].
Acid precipitation operates by a somewhat similar mechanism in the sense that it helps pull ordered water away from the surface of the protein; however, in this case it is due to hydration of the salts present in solution. The end result of this phenomenon is by contrast an enhancement of hydrophobic interactions between proteins which causes aggregation [5].
Acid precipitation:
Acid precipitation is well characterised and was initially intriguing because of the variety of acids that can be used to facilitate this process. It is also commonly used as a modifier in acetonitrile organic precipitations that function to protonate the carboxyl groups of the proteins, thus excluding acetonitrile which increases the solubilising effect for nonpolar groups leading to improved recovery [7]. The options considered in this work are 10% TCA and 6% HClO4
. In both cases 250 µL of the
acid was added to 500 µL of whole blood diluted with 500 µL of water.
In both of these cases (Figures 3 and 4), the supernatant that was produced was clear but also contained deposits and other cellular materials that stuck to the sides of the tube post centrifugation. The perchloric acid being a stronger ‘super acid’ resulted in more pronounced cellular deposits.
Water miscible organic precipitation:
Mixtures of acetonitrile and methanol – a tradeoff between efficient precipitation, and analyte solubility.
It has been shown that acetonitrile does a better job of precipitating proteins out of solution than methanol [5]. This is most likely due to acetonitrile’s ability to more efficiently remove ordered water as its triple bond pi-stacks with cationic and aromatic moieties on the protein’s surface [6]. In addition, as an aprotic solvent it will readily accept and hydrogen bond with free waters. However, as it is noted previously, adding acid to a acetonitrile precipitation can improve recovery of compounds [7] and methanol’s role as a protic solvent most likely contributes to the same effect, helping to improve the solubility of analytes of interest and boost recovery of hydrophobic compounds. It is for that reason that 10:90 (v/v), 50:50 (v/v) and 90:10(v/v) ACN:MeOH were investigated.
After the osmotic breakdown was performed by using a 1:1 dilution with water and light vortexing, a 3:2 ratio of organic to sample was tested while varying the ratio of acetonitrile to methanol as precipitating reagents which has been previously shown to effectively remove proteins from human plasma samples [5]. Figure 5 shows that the increased amount of acetonitrile, 90:10 (v/v) ACN:MeOH yields a brighter red colour in comparison to the methanol precipitate. This suggests that acetonitrile does a better job releasing haemoglobin and potentially any drug bound analytes into the liquid part of the sample. This is supported by previous work that states that haemolytic and non-haemolytic samples can be easily differentiated by their colour but should be confirmed via UV [8]. However, even complete haemolysis does not necessarily ensure that the analytes are totally broken up from the crashed out proteins, which can impact recovery.
Figure 5: ACN:MeOH (90:10) v/v vs. ACN:MeOH (10:90) v/v precipitation of whole blood [1].
Figure 3: TCA precipitation post centrifugation (left) and Figure 4: HCl04
post centrifugation (right) [1].
In addition to producing a brighter red colour, the 90:10 (v/v) ACN:MeOH precipitate also forms a clearer supernatant as seen in Figure 6.
ZnSO4 + organic solvent:
The use of an inorganic salt
such as ZnSO4 and organic precipitating reagents were also evaluated. As seen in Figure 7, these results follow the trend described in the previous section (organic precipitation), where a higher concentration of acetonitrile yielded brighter red samples, and correspondingly clearer supernatant Figure 8.
Figure 6: 90:10 (v/v) ACN/ MeOH precipitation of whole blood [1].
Figure 7: ZnSO4 (left) vs. ZnSO4
MeOH ACN (right)
precipitation of whole blood [1].
Figure 8. ZnSO4+ (left) vs ZnSO4
ACN (right)
precipitation of whole blood [1].
Post Dilution Comparison
To prepare the sample for SPE, pretreatments that influence cation- exchange and reversed phase SPE cleanup had to be considered. Acidifying the sample with 0.1% formic acid ensures that all bases are ionised and able to interact with the sulphonic acid cation-exchange moiety. The aqueous dilution also serves a second purpose which is to dilute the organic solvent making it more amenable for reversed phase interaction. However, it serves neither of these purposes in the context of the acid precipitation as the sample is already acidic and 100% aqueous.
Both the organic precipitating reagents with osmotic breakdown and the ZnS04
lysed
cells with acetonitrile showed some degree of turbidity after dilution (Figures 9 and 10), which may imply that further cleanup is necessary. This most likely occurred for one of two reasons, the formic acid caused further protein precipitation in the
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