1. Recycled mobile phase could contaminate my samples during the chromatographic run.

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1 Page 1 of 5 1. Recycled mobile phase could contaate my samples during the chromatographic run. This is a reasonable concern especially in a regulated laboratory environment. At first this might seem like you would be contaating the mobile phase and generating spurious peaks in the chromatogram, but closer exaation of the process will expose the error of this assumption. Let s see the worst case scenario: When mobile phase is recycled without a recycler apparatus, the sample components leaving the detector are sent to the mobile phase receiver contaating your mobile phase. The level of mobile phase contaation will be affected by the following factors: Sample composition Sample component concentration Injection volume Mobile phase reservoir volume Component response factor Method LOD Since the amount of sample injected in the mobile phase is very small and also diluted in the mobile phase reservoir (>1 L), generally its concentration in the mobile phase remains below the method LOD and it won t be detected. It is necessary to make multiple injections before component concentration in the mobile phase reaches the LOD established by the method. Let s see the next example: Chromatographic conditions: Sample Inj. Volume = 1 µl Mobile Phase Volume = 4 L Sample concentration = 5 mg/l LOD = 1. mg/l 1 µl x 5 mg x 1 L x 1 =.5 mg/inj 1 inj 1L 1 ml 1 µl.5 mg =.125 mg < 1. mg/l (LOD) 4 L 1 L.125 mg x 32, inj = 4 mg = 1 mg/l (LOD) 4 L

2 Page 2 of 5 As we have seen in the above example, it is necessary to make approximately 32, injections before the sample response in the mobile phase equals the method LOD. In this scenario the mobile phase was recycled without a recycler system. Can you imagine the results using a mobile phase recycler system which is designed to re-use only the uncontaated mobile phase generated during the chromatographic run? The probability to contaate the sample using the mobile phase recycler would be close to zero. Note: An errors, normally accepted in some chromatographic methods, causing more contaation in the sample chromatogram than the one associated to the mobile phase recycling process is the injection carry over. Also, the system suitability test, which could include a mobile phase sample injection, can be used to detere if the mobile phase is contaated and suitable for your analysis before the sample is injected. 2. Accuracy of the sample results can be affected by the use of the mobile phase recycler. As mentioned before, mobile phase contaation using a mobile phase recycler system has low probabilities and can be easily detected by the system suitability test before the analysis. But if for any reason the mobile phase is contaated the standard solution injected during the chromatographic run would correct any error associated with it. Therefore accuracy of the sample results would not be affected by the contaated mobile phase. There are two HPLC errors, normally accepted in some chromatographic methods, causing more accuracy errors than the one associated to the mobile phase recycling process: HPLC injector error Injection carry over error Note: Both errors are based in the HPLC design, HPLC maintenance and the chromatographic method.

3 Page 3 of 5 3. Regulatory observations can be generated in regulated laboratories with the use of mobile phase recycler systems. As other equipments within the regulated laboratory, mobile phase recycler system should be validated according to the user requirements and should be used according to an approved procedure before use. Generally the following qualification tests are required for this type of instrument: Installation Qualification Operation Qualification Performance Qualification o Since each chromatographic method has different peak shape and size, the PQ should be performed by each method. o Expiration date of the re-used mobile phase should be detered for each method. o In a regulated laboratory the following procedure should be prepared: Operational procedure Maintenance procedure Calibration procedure No internal or external observation will be generated if mobile phase recycler system is properly validated and used according to an approved procedure. 4. What is the best mobile phase recycler in the market today? Traditional mobile phase recycler systems use a fixed voltage level (or threshold above baseline) which must be exceeded before a peak can be detected. Consequently the following situations can be observed: Clean Mobile Peak Clean Mobile A positive baseline drift could send clean mobile phase to waste instead of sending it to the mobile phase reservoir.

4 Page 4 of Peak A negative baseline drift could send contaated mobile phase to the mobile phase reservoir instead of sending it to waste Smaller peak not detected and peak tailing could send contaated mobile phase to the mobile phase reservoir instead to sending it to waste.

5 Page 5 of 5 Change in the baseline noise could affect the recycler efficiency. I recommend the SolventTrak because is the only recycler designed to eliate components (peaks) from the used mobile phase even if chromatographic run has positive or negative baseline drift. [] SolventTrak - 4_23_28 8_4_41 PM - ST Signal SolventTrak - 4_23_28 8_4_41 PM - Valve Signal 1 5 Voltage Time [.] SolventTrak performance during a chromatographic run with normal, negative, and positive baseline line drift.