Evaluation of linear and step gradient performance and ...

1 Evaluation of linear and step gradient performance and retention time precision of the Agilent 1200 Series Rapid Resolution LC. This document is believed to be accurate and up-to-date. However, Agilent Technologies, Inc. cannot assume responsibility for the use of this material. The information contained herein is intended for use by informed individuals who can and must determine its fitness for their purpose. Evaluation of linear and step gradient performance and retention time precision of the Agilent 1200 Series Rapid Resolution http:// Page 1 of 1. Evaluation of linear and step gradient perfor- mance and retention time precision of the Agilent 1200 Series Rapid Resolution LC. system Technical Note 3 4 5 min Introduction A main performance criterion of applications is evaluated.

2 Further, any LC system is precision of based on tracer experiments, it retention times, which is influ- is demonstrated that linear and enced primarily by the pumping stepwise gradients are delivered device. It is important that the with excellent accuracy and selected flow is delivered precise- precision. The experiments also ly and that for gradient operation show that the variability of the the mobile phases are mixed delay volume meets the demands reliably over the complete of applications using narrow-bore gradient range. In this Technical columns as well as standard-bore Note the precision of retention columns. times for gradient and isocratic Experimental Equipment An Agilent 1200 Series Rapid Res- olution LC system was used with the following modules: Agilent 1200 Series binary pump SL with vacuum degasser for applications using m parti- cle columns up to 150-mm length and with internal diame- ters from to mm Agilent 1200 Series high-perfor- mance autosampler SL for high- est area precision Agilent 1200 Series thermostat- ted column compartment SL.

3 With wide temperature range from 10 degrees below ambient up to 100 C. Agilent 1200 Series diode-array Figure 1. detector SL for 80-Hz operation, Standard and low delay volume configurations of the Agilent 1200 Series binary pump SL. including new data protection tool suited for applications where low- path. To change to the low delay ZORBAX SB C-18 columns with est noise and highest resolution volume configuration, the flexible different internal diameters and for optimum quantification is id capillary between the lengths, packed with m needed. For applications where a mixer and the upper position of particles low delay volume is needed the the pressure sensor is disconnect- damper and the mixer can be ed. Then, the capillary originally Pump design switched out of the flow path, attached to the mixer is connected The Agilent 1200 Series binary resulting in a delay volume of to the pressure sensor.

4 The pump SL is designed to meet the 120 L. This configuration is best damper and mixer are now not in demands of high throughput and suited for application where the flow path. Degassing is recom- highest performance for optimum speed and lowest peak dispersion mended for all application ranges resolution and lowest pump ripple are needed. To enable these to ensure optimum performance . for a wide range of applications changes the pressure transducer The Agilent 1200 Series micro on narrow or standard-bore is separated from the damper. degasser SL has 4 channels and an columns. Figure 1 shows a Both pump heads have an addi- internal volume of 1 mL. Each schematic of the 1200 Series binary tional damping coil each of 500 L channel degasses the solvents pump SL.

5 The delay volume can volume and an additional outlet continuously. The following sec- be configured according to the valve. This does not add delay tions discuss the performance of application needs. The standard volume during gradient analysis both pump configurations; with configuration is best suited for because mixing occurs later in and without mixer and damper in highest performance in terms of the flow path. The pump can be the flow path. The configuration noise and resolution. In this stan- used up to pressures of 600 bars, with mixer and damper in the flow dard configuration the mixer and providing for the use of sub-2- path delivers optimum resolution damper are in the flow path and micron particle columns up to and lowest pump ripple and is the delay volume is between 600 150-mm length.

6 In the standard best suited for the analysis of and 800 L, depending on the delay volume configuration the complex samples where highest pressure. This configuration is mixer and damper are in the flow 2. resolution and lowest system DAD1 A, Sig=245,10 Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\ ). DAD1 A, Sig=245,10 Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\ ). DAD1 A, Sig=245,10 Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\ ). noise is needed. The configuration DAD1. DAD1 A, A, Sig=245,10. Sig=245,10 Ref=360,80. Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\ ). (E:\CHEM32\1\DATA\INJECTIONVOL2\ ). DAD1 A, Sig=245,10 Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\ ). DAD1 A, Sig=245,10 Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\ ).

7 Without damper and mixer in the DAD1. DAD1 A, A, Sig=245,10. Sig=245,10 Ref=360,80. Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\ ). (E:\CHEM32\1\DATA\INJECTIONVOL2\ ). DAD1 A, Sig=245,10 Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\ ). flow path is best suited for LC/MS mA. Overlay of 10 runs or LC/UV using ID columns. 25. Injection volume: 50 L. Injected amount: 80 ng Retention time precision of Agilent RSD RT < %. 20. 1200 Rapid Resolution LC system for gradient LC analysis Retention time precision was 15. evaluated for both pump configu- rations under gradient and isocrat- 10. ic conditions using standard bore and narrow bore columns. In the first example the standard delay 5. configuration was tested using a standard bore phenone 0.

8 Mix was analyzed using gradient 1 2 3 4 5 min conditions from 35 to 95 %. A. 100 x mm column and a flow rate of mL/min were selected. Figure 2. Overlay of 10 chromatograms. These conditions are close to con- ditions used in conventional LC. analysis. Figure 2 shows 10 runs overlaid, demonstrating the reten- Chromatographic conditions: Test sample: Set of 9 compounds, 100 ng/uL each, dissolved in water/ACN (65/35). tion time precision obtained for 1. Acetanilide, 2. Acetophenone, 3. Propiophenone, 4. Butyrophenone (200 ng/mL), this application. The run time was 5. Benzophenone, 6. Valerophenone, 7. Hexanophenone, 8. Heptanophenone, 6 min. The Evaluation of the reten- 9. Octanophenone Column: 100 x mm ZORBAX SB C-18, m for 600 bar operation tion time precision was found to Pump: Solvent: A = Water, B = ACN.

9 Be less than % RSD. gradient : 35 to 95 %B in 5 min, hold at 95 %B for 1 min Stop Time: 6 min Post Time: 2 min Flow Rate: mL/min Autosampler: Injection volume: 50 L. Wash 10 s for exterior of needle Thermostatted column compartment: Temperature: 50 C. Detector: 13 L cell 20-Hz data acquisition Peak width = < min Slit: 8 nm Signal: 245/10 nm, ref 360/80 nm 3. Retention time precision of Agilent 1200 Rapid Resolution LC system %RSD, SD= min Limit QA/QC = %RSD, SD= min using the low-delay volume configu- %RSD, SD= min %RSD, SD= min ration RT Precision (n=10). In the previous section the reten- Flow= mL/min, T=40 C. tion time precision of the stan- Flow= mL/min, T=80 C. dard delay configuration was eval- Flow= mL/min, T=40 C.

10 % RSD. uated. In this section the retention Flow= mL/min, T=80 C. time precision of the low volume delay configuration will be evalu- ated. The analyses were run at high and low flow rates as well as e ph ne at high and low temperatures. For e e e ne ne ne on an non on on no no op eno o en Ac ilide Be hen Bu hen Va hen Oc phe all low flow rate applications the e He phe He oph h an p p op op o no ro io ro n et relative standard deviation for the et nz ty ta le Average RSD. xa Ac pt Pr retention times was typically less than %. For high flow rate and ultra-fast applications the Figure 3. relative standard deviation for the Statistical Evaluation of the retention time precision for the repeated analysis of a nine-com- retention times was typically less pound mixture under various conditions using the low delay configuration of the Agilent 1200.