Standardized Ion Chromatograpy (IC) for Salivary Analysis

Standardized Ion Chromatograpy (IC) for Salivary Analysis According to Darvell et. al. (2004), the research is about analysis of human saliva by using ion chromatography(IC). The aim of this research is to construct out a standardized IC protocol for future salivary analysis. The samples has go through several procedures such as centrifugation to remove particulates, filtration on supernatant, and the residual droplet was removed by a paper tissue without touching it to the CFD (centrifugal filter device). This is because want to minimize the risk of back–contamination of the device. About the pretreatment, the temperature for centrifugation is set at 4Â °C which inhibit the pathogen spreading and lower the bacterial activity. The steps of pretreatment temperature, duplicate runs of the samples and recovery of known addition of standard solution are to make sure that the result obtained is reproducible and no significant different. However, the resolution has shown decrease and this may cause by the protein retained in the exchange site of column resin or the surface of suppressor. It is suggested that application of membrane ultrafiltration can minimize contamination of IC system. Besides, there are few things have to take in consideration which are pretreatment and sample dilution. If the sample pH is higher than 2-3, in pretreatment part have to avoid introducing the ionic contaminant or subtracting any ion that needed if cellulose used in material for membrane ultrafiltration. Sample dilution can make the resolution between peaks greater especially when fluid with smaller volume are available sufficient for injection. Xie et al. (2011) have reported the studies on levels of adenosine phosphate in blood which are analyzed by using ion chromatography. In this research, gradient mode of elution is used, because it well-known as can carry out fast result and provide sharp peak due to gradient compression effects. The procedure are firstly sample dilution, homogenization, centrifugation, membrane filtration and finally injection into the IC system. The addition of acetonitrile in homogenization part is to extract out the protein as precipitation. Adenosine phosphate likely retained in column and cause contamination since they are very polar molecule however this research has proven that by using this method it will not retained on the column and it can applicable to the analysis of true blood samples. Hou Cramer (2011) has evaluated on selectivity in multimodal anion exchange system priority on prediction of protein retention time and investigation of effect caused by mobile phase modifier. About selectivity, as we know protein are more strongly retain tendency with resin. The multimodal effect which the arrangement pattern has given an electrostatic attraction that manage the binding behaviors with obeying Langmuir isotherm. In this research, two anion resin was used which are Capto Adhere(ADH) and Q Sepharose FF. Result shown there was more retention on ADH because additional interactions existed. In ADH ligand, the additional interaction may composed hydrophobic properties contributed by aromatic ring and hydrogen bonding contributed by hydroxyl group. Suggested that the size of ion-exchanger should be smaller to optimize the performance and using monolithic porous column in order to improve the morphology of the resins. According to the Tyrrell ed. al. (2009), rapid IC have several advantages such as reduce the analysis time, cost and improve the productivity. The rapid IC can be transfer from the conventional method by modifying some parameter. The rapid IC using the similar stationary phases with the conventional chromatographic but the column geometry used in rapid IC was modified. Besides that, the volume used and the flow rate of sample were reduce by a factor of 12 to speed up the chromatographic process and the resolution is not affected for the smaller particle-packed columns with shorter length. In generally, gradient elution are consider more beneficial in fast separations and multi-component sample analysis. In optimize conditions, the separation speed is assume constant in this study. Meanwhile, the other parameter such as the column length was decreased by a factor of 10 and the smaller particles were reduced by a factor of 15. Reductions by factor in the parameters was reduce the resol ution, but the reduction in resolution still in the acceptable ranges, so the process still can be accepted. According to Chen Leong (2009), ÃŽ ±-fetoprotein (AFP) is a potential candidate in biopharmaceutical field, especially in the treatment of autoimmune indications. Recombinant AFP can indicate rheumatoid arthritis recently. However, due to the complexity of its structure as it contains 32 cysteines, which refolding and form 16 disulfide bonds by oxidation; the refolding process will be very hard to occur because the protein with highly disulphide-bonding is immobile on a stationary phase of chromatography. The objective of this study was to improve the refolding yield of the recombinant human AFP (rhAFP) using anion-exchange chromatography. Refolding process by batch dilution was studied as a control. Instead of ion-exchange column, a PD-10 desalting column was used to remove the DTT from protein mixture. The mixture was then eluted out the desalting column and refolding buffer was added. There were two types of anion-exchange column to carry out on-column refolding, which were Q-FF columns and DEAR-FF columns. Same procedures were performed on this refolding process as batch dilution, the protein was incubated in the column with refolding buffer for 0-24 hours, but with an addition of stripping buffer after refolding cycle. Next off-column refolding was carried out using a 1ml Q-FF column. Unlike on-column refolding, the protein was not incubated in column but treated with refolding buffer with or without 0.5M L-arginine after elution process. For batch dilution, as a result, the refolding yield is at maximum level which was around 55% at less than 0.5mg/ml of rhAFP, but at the allowable greatest concentration of protein refolding which is 0.45mg/ml, the yield of refolding obtained is 14% only. This is because high concentration of protein would lead to high aggregation and hence, low refolding process. 0.45mg/ml was the highest protein refolding concentration in this section because of solubility and viscosity limitation in preparation step of higher concentr ation of protein sample. On the other hand, refolding process in DEAE-FF column had higher recovery yield of rhAFP compared to that in Q-FF column which were 19% and 15% respectively when the amount of rhAFP load was 1mg. Q-FF is a strong anion-exchanger compared to DEAE-FF, this caused the strong interaction between protein samples with matrix and limited the disulfide shuffling to refold. In contrast, the interaction in DEAE-FF is relatively weaker, so the disulfide shuffling had higher efficiency and increased refolding process occurrence. The stripping buffer was added after refolding cycle to elute more refolded rhAFP, as the protein was mostly bound on column during refolding and elution process. Similar to result of batch dilution, increased amount of protein load brought increased aggregation and lower refolding process. Furthermore, aggregation of protein at the top section of the column can prevent the protein to move through the column, so another test was done with addit ion of NaCl to refolding process conducted in DEAE-FF column and it showed higher refolding yield. Investigation of off-column refolding was done without incubation of protein in Q-FF column with refolding buffer. Without addition of L-arginine, the refolding yield of 1mg of rhAFP load was higher than on-column refolding, and the yield could be increased to 42% after addition of L-arginine. The possible reason is due to interference of L-arginine toward the adsorption of rhAFP on the matrix. Hence it is impossible to add L-arginine if on-column refolding was desired to be performed. Actually, instead of Q-FF column, the off-column refolding can be conducted on DEAE-FF column with addition of L-arginine and NaCl, the outcome should be interesting to investigate. In a nutshell, off-column refolding yielded the most among the methods, which was 13-fold increase from batch dilution.