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Principles of Operation Sample Position in the Sample Loop Ideally, the injection volume is injected exactly into the sample loop. In reality, the injection volume is often slightly offset. Therefore, the front and rear volume are used to ensure the loop is full with sample. This is illustrated schematically in Figure A-16, where: • Blue—air (front air gap and rear air gap) • Yellow—sample (rear volume) • Orange—sample (injection volume) • Pink—sample (front volume) Even when the injection volume is not located exactly in the sample loop, the presence of the front and rear volume ensures that the entire volume injected onto the column contains sample. Figure A-16 Possible Locations of Sample in Loop, Front and Rear Volume >0 Sample Loop Injection volume exactly in loop Injection volume offset, rear volume follows sample Injection volume offset, front volume precedes sample Flow Because the front and rear volumes can be set to 0 by the user in the autosampler method in the Analyst® software, it is possible that the volume injected onto the column can include an air bubble. This is illustrated schematically in Figure A-17, where blue represents air and orange represents the injection volume. Figure A-17 Possible Locations of Sample in Loop, Front and Rear Volume = 0 Sample Loop Injection volume exactly in loop Injection volume offset, air bubble follows sample Injection volume offset, air bubble precedes sample Flow Communicating with the DLW The Analyst® software uses the autosampler method to communicate with the HTC-xt PAL autosampler and the DLW. The autosampler method is part of the acquisition method. The Available Cycles field in the CTC PAL Autosampler Basic Properties tab of the Acquisition Method window (Figure A-18) lists the available autosampler methods. The appropriate method for the HTC-xt PAL autosampler is “microLC200-Injection-Rev B” and is installed with the Eksigent MicroLC 200 Plus system. Eksigent MicroLC 200 Plus System 114 of 140 Operator Guide RUO-IDV-05-1129-B | D5033433 G