Split elution

A standard split elution technique is commonly employed which allows each portion of eluting agent to carry out two elutions and thus achieve a high uranium concentration. 

The feed solution is prepared by acid leaching, removal of gross solids by filtration or counter-current decantation, together with a final polishing filtration step, often in a sand-bed clarifier. Using such a feed, a typical cycle of operations for a four-column system with split elution is as follows ... 

After the main part of the vanadium has been eluted, the remainder is removed by long contact time of 30 to 45 min on the bed. A total of four column volumes of eluate are necessary and the column is then washed with two volumes of very dilute sulphuric add. A split elution technique has been developed which gives vanadium concentrations of up to 100 g/1. in the eluate. Purification from some impurities, notably iron, appears to be fairly good, although a special primary elution stage for iron with 0 9N ammonium chloride and 0-lN hydrochloric acid can be introduced before vanadium elution when the iron content is high. A few per cent of the vanadium may be lost during the iron elution. 

We use the second-dimension separation from Fig. 6.6 with a 25 pL injection volume and 2.5 min sampling time the separation is an RPLC method that uses a monolithic column. Thus, 10 pL/min is the maximum flow rate in the first-dimension. Fig. 6.7 shows the development of the first-dimension column that utilizes a hydrophilic interaction (or HILIC) column for the separation of proteins at decreasing flow rates. The same proteins were separated in Fig. 6.6 (RPLC) and 6.7 (HILIC) and have a reversed elution order, which is known from the basics of HILIC (Alpert, 1990). It is believed that HILIC and RPLC separations are a good pair for 2DLC analysis of proteins as they appear to have dissimilar retention mechanisms, much like those of NPLC and RPLC it has been suggested that HILIC is similar in retention to NPLC (Alpert, 1990). Because the HILIC column used in Fig. 6.7 gave good resolution at 0.1 mL/min and no smaller diameter column was available, the flow was split 10-fold to match the second-dimension requirement... 

The loop for the 2nd-D was loaded with the effluent of the 1 st-D at 50 pL/min for 1 min 58 s, and then the injection valve was turned to inject the 100 pL fraction for 2 s onto the 2nd-D HPLC. The flow rate was 5 mL/min, and the valve was turned back for the next loading, resulting in fractionation of the lst-D every 2 min. In this case less than 2% of the effluent from the 1 st-D was wasted during sample injection. The 2nd-D effluent eluted at 5 mL/min from the 2nd-D column, passed through a UV detector, and then was split by using a T-joint at approximately a 1/140 split ratio, resulting in a flow rate of ca. 36 pL/min going into the spray capillary for ESI-TOF-MS detection. 

Using the Tomtec Quadra 96 workstation, 0.1 mL of the ethyl acetate layer was transferred to a 96-well collection plate containing 0.4 mL of acetonitrile in each sample well. The solution was mixed 10 times by aspiration and dispersion on the Tomtec. The plate was then covered with a sealing mat and stored at 2 to 8°C until LC/MS/MS analysis. The HILIC-MS/MS system consisted of a Shimadzu 10ADVP HPLC system and Perkin Elmer Sciex API 3000 and 4000 tandem mass spectrometers operating in the positive ESI mode. The analytical column was Betasil silica (5 fim, 50 x 3 mm) and a mobile phase of acetonitrile water formic acid with a linear gradient elution from 95 5 0.1 to 73.5 26.5 0.1 was used for 2 min. The flow rate was 1.0 mL/min for the API 3000 and 1.5 mL/min for the API 4000 without any eluent split. The injection volume was 10 jjL and a run time of 2.75 min was employed. 

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