Performance Characteristics of the Agilent 1260 Infinity II Bio-Inert LC

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1 Performance haracteristics of the gilent 16 Infinity II io-inert L Technical Overview uthors Sonja Schneider and Uschi Schweikert gilent Technologies, Inc. Waldbronn, Germany Introduction The gilent 16 Infinity II io-inert L is the next generation of gilent io-inert L, specially designed for conditions used in biochromatography (high salt concentrations such as M Nal, up to 8 M urea, and high and low ph solvents such as. M NaOH or. M Hl) by working with a completely inert sample flowpath. ll capillaries and fittings throughout the multisampler, multicolumn thermostat, and detectors, are metal free, and biomolecules come in contact only with ceramics or PEEK. esign elements in the gilent 16 Infinity II io-inert Multisampler and gilent 16 Infinity II io-inert Multicolumn Thermostat (MT) provide the user with the highest flexibility. ased on the proven technology of the gilent 16 Infinity Series liquid chromatography platform, the 16 Infinity II io-inert L has equivalent performance specifications as the standard 16 Infinity io-inert Quaternary L 1. The 16 Infinity II io-inert L is compatible with standard methods, with a maximum pressure of 6 bar.

2 The 16 Infinity II io-inert L offers: Flow rate range up to 1 ml/min Maximum pressure of 6 bar for UHPL. Higher pressures allow support of either higher flow rates (more speed) or longer columns (more resolution). The gilent 16 Infinity II io-inert Multisampler is based on a proven flow-through design using a ceramic needle. Further elements are the sample hotel for various sample containers that hold up to eight vial or microtiter plates, the robotics and needle handling routine for shortest injection cycle times, and the multiwash function for lowest carryover. n advanced column capacity for up to six columns, with individual precolumn solvent heating in a single MT. The door provides superior usability with flexible flap positions at 9 or 18, and it can be removed completely for highest accessibility. The MT facilitates precise column thermostatting for maximum application flexibility. These performances are possible by Peltier cooling and heating with two independent temperature zones from 1 degrees below ambient (minimum 4 ) to 8. In addition, the MT can be equipped with a column tag reading option for automatic and trackable column management. gilent 16 Infinity II L iode rray etector () WR with bio inert standard flow cell Standard-bore UHPL and conventional applications run on the same system configuration. Revolutionary gilent InfinityLab Quick onnect UHPL column fittings for dead-volume-free fluidic connections that enable efficient, fast, and convenient column exchange. In addition, easy-to install precolumn gilent InfinityLab Quick-onnect heat exchangers are available for precolumn solvent heating (Figure 1). Experimental The 16 Infinity II io-inert L that was tested comprised: gilent 16 Infinity II io-inert Pump (G64) gilent 16 Infinity II io-inert Multisampler (G668) with sample cooler (Option #1) gilent 16 Infinity II Multicolumn Thermostat (G7116) with bio-inert heat exchanger (Option #19) gilent 16 Infinity II iode rray etector WR (G711) with bio inert flow cell (Option #8) Figure 1. gilent InfinityLab Quick onnect fittings and gilent InfinityLab Quick-onnect heat exchangers.

3 Pump performance retention time (RT) precision The most important parameter influencing retention time (RT) precision is pump performance. RT precision was tested with different gradient and isocratic conditions using 4.6 and.1 mm id columns. The relative standard deviation (RS) of RTs for conventional gradient runs was <.4 %RS, except for the first peak for n = 7 (Figure.) For fast gradients, with a run time of approximately 1 minute, the RS for RTs was <.1 %RS (Figure 3) for n = Peak I RS RT (%) 1. Uracil.113. Phenol Methyl paraben Ethyl paraben.8. Propyl paraben n,n-iethyl-m-toluamide utyl paraben. 8. Toluene Heptyl paraben Sample from Sigma-ldrich: Reversed Phase Test Mix, Order No.: U 1 1 ml (uracil, phenol, n,n-diethyl-m-toluamide, toluene) HPL Gradient System iagnostic Mix, Order No.: ml (phenol, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, heptyl paraben, uracil) Sample preparation ilute each sample to ml with water/acetonitrile 1:1 Mix the two diluted samples 1:1 olumn gilent ZORX S 18, 4.6 mm 1 mm, µm Mobile phase ) Water, ) cetonitrile Gradient minutes %, 1 minutes 9 % Flow rate 1 ml/min Stop time 1 minutes Post time minutes Injection volume µl olumn temperature 3 4/4 nm; Ref 4/1 nm io-inert standard flow cell, 1 mm Peak width <. minutes (1 Hz) Figure. Precision of RTs for conventional gradient runs Peak I RS RT (%) 1. cetophenone.89. Propiophenone utyrophenone Valerophenone.. Hexanophenone Heptanophenone Octanophenone.9 8. enzophenone cetanilide Sample: RRL heckout sample (p/n ) cetophenone, propiophenone, butyrophenone, valerophenone, hexanophenone, heptanophenone, octanophenone, benzophenone, acetanilide olumn gilent Poroshell 1 E 18,.1 mm mm,.7 µm Mobile phase ) Water, ) cetonitrile Gradient minutes 3 % 1 minute 9 % Flow rate ml/min Stop time 1. minutes Post time 1 minute Injection volume 1 µl olumn temperature 4/1 nm; Ref 36/1 nm io-inert standard flow cell, 1 mm Peak width <.6 minutes (4 Hz) Figure 3. Precision of RTs for fast gradient runs. 3

4 Figure 4 shows conventional isocratic conditions with an RT precision of <.6 %RS for n = 7. omparison of an gilent 16 Infinity io-inert Quaternary L and an gilent 16 Infinity II io inert L step gradient Tracer experiments are frequently used to verify the solvent mixing ripple at different gradient mixtures to evaluate pump performance. Figure shows a step gradient from to 1 % in 1 % steps with caffeine as the tracer compound. n overlay of step gradients generated with the 16 Infinity io-inert Quaternary L and the 16 Infinity II io-inert L is shown. The performance of both systems is highly comparable regarding the mixing properties of the pump. 1,7 1,6 1, 1,4 1,3 1, 1,1 1, Peak I RS RT (%) RS rea (%) 1. imethylphtalate iethylphtalate iphenyl o-terphenyl Isocratic sample (p/n ) olumn gilent ZORX S 18, mm, 1.8 µm Mobile phase ) Water, ) cetonitrile Isocratic 7 % Flow rate 1. ml/min Stop time 9 minutes Injection volume µl, draw speed µl/min olumn temperature 4 4/4 nm; Ref 38/1 nm io-inert standard flow cell, 1 mm Peak width <. minutes, (1 Hz) Figure 4. RT precision for conventional isocratic runs gilent 16 Infinty io-inert Quaternary L, 149 bar gilent 16 Inffinity II io-inert L, 13 bar olumn Restriction capillary Mobile phase ) Water + % isopropanol ) Water + % isopropanol + 1 mg/l caffeine Step gradient From to 1% in 1% steps Flow rate 1 ml/min Stop time minutes olumn temperature 36 73/4 nm; Ref 38/1 nm; peak width: <.13 minutes, ( Hz) io-inert standard flow cell, 1 mm Figure. Overlay of step gradients. gilent 16 Infinity io-inert Quaternary L and an gilent 16 Infinity II io-inert L. 4

5 Low backpressure application protein separation by size exclusion chromatography (SE) at 1 bar Low backpressure applications, commonly used in protein analysis, were tested with the 16 Infinity II io inert L to prove pressure stability and precision of RT with the 6 bar system. High precision of RTs was obtained (RS of <.4 %, n = ). Figure 6 shows a separation of bovine serum albumin (S) from its dimer using an SE column with a low backpressure, resulting in a total system pressure of 1 bar. Injector performance area precision Precise injection is mandatory for good quantitative results in liquid chromatography. The 16 Infinity II io-inert Multisampler can inject precisely over an injection range of. to 1 µl. Figure 7 shows an example chromatogram for an injection volume of 1 µl. The RS was <.37 % for n = 7. The RS for an injection volume of. µl was <.4 % for n = S imer 13 ka S 66 ka olumn SE, 1 3 mm, 13 µm Mobile phase ) Phosphate buffered saline, ph = 7. Isocratic 1 % Flow rate. ml/min Stop time 6 minutes olumn temperature Room temperature 8/4 nm; Ref 36/1 nm; peak width: <.1 minutes, (. Hz) io-inert standard flow cell, 1 mm RS of RT in % S.43 S imer.16 Figure 6. Low backpressure application: separation of bovine serum albumin (S) at 1 bar Peak I RS RT (%) RS rea (%) 1. cetophenone Propiophenone utyrophenone Valerophenone Hexanophenone Heptanophenone Octanophenone enzophenone cetanilide Sample: RRL heckout sample (p/n ) cetophenone, propiophenone, butyrophenone, valerophenone, hexanophenone, heptanophenone, octanophenone, benzophenone, acetanilide olumn gilent Poroshell 1 E 18, 3 mm,.7 µm Mobile phase ) Water, ) cetonitrile Gradient minutes %, 8 minutes 8 % Flow rate 1. ml/min Stop time 8 minutes Post time 4 minutes Injection volume 1 µl olumn temperature 3 4/1 nm; Ref 4/1 nm io-inert standard flow cell, 1 mm Peak width <. minutes (1 Hz) Figure 7. rea precision for conventional gradient runs for 1 and. µl.

6 Figure 8 shows an example chromatogram for an injection volume of µl. The RS is <.14 % for n = 7. The injector settings are important for optimum precision of areas. If the highest precision is needed, the draw speed of the injector should be set to lower values, especially if large volumes or highly viscous samples are injected. It is important to avoid solvent evaporation out of the sample vials, and decomposition problems using a cooled autosampler. Injector performance carryover For the injection, the draw speed was set to µl/min, and an exterior needle wash for 1 seconds was used (Figure 9). No carryover was detected for the conditions used. fter a 1,-ng sample injection, unadulterated solvent was injected. Recommendations for carryover and cleaning procedures Flush port wash solvent must always be installed and used. The solvent chosen should be able to dissolve the sample compounds. It is highly recommended to reconnect the capillary connections from time to time to prevent cavities, which can lead to enhanced carryover. 1,7 1,6 1, 1,4 1,3 1, 1,1 1, Peak I RS RT (%) RS rea (%) 1. imethylphtalate iethylphtalate iphenyl o-terphenyl Isocratic sample (p/n ) olumn gilent ZORX S 18, mm, 1.8 µm Mobile phase ) Water, ) cetonitrile Isocratic 7 % Flow rate 1. ml/min Stop time 9 minutes Injection volume µl, draw speed µl/min olumn temperature 4 4/4 nm; Ref 38/1 nm io-inert standard flow cell, 1 mm Peak width <. minutes, (1 Hz) Figure 8. rea precision for isocratic runs with µl injection volume. 1, ,8 1. 1,7 1, ng 1,6 hlorhexidine 1. 1,.8 1,4.6 1,3.4 No carryover detected 1, 1,1. 1, Sample: hlorhexidine 1 mg/ml dissolved in.1 % TF in H O dd olumn gilent Poroshell 1 E 18, 4.6 mm mm Mobile phase ).1 % TF in in H O dd ).1 % TF in cetonitrile Isocratic 4 % Flow rate.6 ml/min Stop time. minutes Injection volume 1 µl, draw speed µl/min 1-second needle wash (exterior) olumn temperature 4/4 nm; Ref 36/1 nm io-inert standard flow cell, 1 mm Peak width <.1 minutes, ( Hz) Figure 9. No carryover was detected after injection of 1, ng chlorhexidine. 6

7 Injector performance injection volume linearity Injection volume linearity was tested using caffeine standards. ll injection volumes contained ng of caffeine. s a result, the injection volume was varied, but the injected amount always remained the same (Figure 1). The peak heights and areas should be the same for all injection volumes. The experiments showed that all areas were within 1.4 % RS over the complete injection volume range of.78 to 1 µl for n =. Performance of the gilent 16 Infinity II WR The 16 Infinity II Wide Wavelength Range (WR) with the bio inert standard flow cell is recommended for all common applications. etector performance linearity Linearity was tested using caffeine standards from 1. to 1,6 ng injected with n =. Good linearity was obtained at this concentration range. The coefficient of correlation was The response factors were within the % error range over an absorbance range of 1.7 to,36 (Figure 11). ll areas were within 1.4 %RS.78 µl 1.6 µl 3.13 µl 6. µl 1. µl µl µl 1 µl Sample: affeine 1 mg/ml, 7 times 1: diluted olumn gilent ZORX Eclipse Plus 18, mm, 1.8 µm Mobile phase ) Water, ) cetonitrile Isocratic 3 % Flow rate.8 ml/min Stop time. minutes Injection volume.78 to 1 µl, draw speed µl/min olumn temperature 4/4 nm; Ref 38/8 nm io-inert standard flow cell, 1 mm Peak width <.1 minutes, ( Hz) Figure 1. Injection volume linearity from.78 up to 1 µl; injected amount was always the same. Response factor (mount/rea) 4.E-1 3.E-1 Height = E-1.E-1.E-1 1.E-1 gilent 16 linearity correlation.9999 Height =, ,6 mount (ng injected) % Range verage response factor =.83 Sample: Enterprise Edition affeine Extended Standards Kit olumn gilent Poroshell 1 E 18, 3. mm, 1.9 µm Mobile phase ) Water, ) cetonitrile Isocratic 1 % Flow rate.8 ml/min Stop time. minutes Injection volume 1 µl, µl (1,6 ng) olumn temperature 3 73/1 nm; Ref 38/1 nm Universal bio-inert standard flow cell, 1 mm Peak width <.1 minutes, ( Hz) Figure 11. Linearity of the gilent 16 Infinity II WR with bio-inert flow cell. 7

8 Multicolumn thermostat comparison of standard and bio inert heat exchangers The column temperature regulation using the bio-inert heat exchanger was compared to the standard (stainless steel) heat exchanger in the MT. Samples were sensitive to temperature changes regarding RT (sulfa drugs: sulfadiazine, sulfathiazole, sulfamerazine, and sulfamethazine). With the change of solvent temperature, component (sulfathiazole) changed position within the order of eluted components during the isocratic run. t 1, component eluted at the third position, whereas at 3 and above it eluted at the second position using the standard heat exchanger (Figure 1) Peak I. Sulfadiazine. Sulfathiazole. Sulfamerazine. Sulfamethazine olumn gilent InfinityLab, E 1, mm,.7 µm Mobile phase ) Water +.1 % TF ) cetonitrile +.1 % TF Flow rate 1 ml/min Gradient minutes, 1 % 1 minutes, % 1 minutes, 1 % Stop time 1.1 minutes Post time minutes olumn temperature 6,, 4, 3,, 1 4/4 nm; Ref 38/1 nm bio-inert standard flow cell, 1 mm Peak width <.1 minutes, ( Hz) Figure 1. Thermosensitive sulfa drugs, with column temperature regulated through the standard heat exchanger. 8

9 Using the bio-inert heat exchanger, no major differences have been seen regarding RT changes (Figure 13). Table 1 shows the relative RT of sulfathiazole (component ) referred to sulfamerazine (component ) with n = 3. oth heat exchangers are highly comparable Peak I. Sulfadiazine. Sulfathiazole. Sulfamerazine. Sulfamethazine olumn gilent InfinityLab, E 1, mm,.7 µm Mobile phase ) Water +.1 % TF ) cetonitrile +.1 % TF Flow rate 1 ml/min Gradient minutes, 1 % 1 minutes, % 1 minutes, 1 % Stop time 1.1 minutes Post time minutes olumn temperature 6,, 4, 3,, 1 4/4 nm; Ref 38/1 nm bio-inert standard flow cell, 1 mm Peak width <.1 minutes, ( Hz) Figure 13. Thermosensitive sulfa drugs, with column temperature regulated through the bio-inert heat exchanger. Table 1. omparison of standard and bio-inert heat exchanger. Relative RT of sulfathiazole (component ) referred to sulfamerazine (component ) Standard heat exchanger io-inert heat exchanger

10 onclusions The performance of the gilent 16 Infinity II io-inert L meets the requirements of modern analytical liquid chromatography. The performance is highly comparable with the gilent 16 Infinity io-inert Quaternary L. The 16 Infinity II io-inert L is well suited for 4.6, 3., and.1 mm id columns, and can be used for conventional HPL and UHPL on columns packed with 1.8 µm particles. Precision of RTs for conventional L is typically <.4 % RS. The precision for peak areas is typically <.14 % for injection volumes > µl. No carryover was detected after 1, ng chlorhexidine injection. References 1. Schneider, S. Performance characteristics of the 16 Infinity io-inert Quaternary L System, gilent Technologies Technical Overview, publication number EN, 11.. gilent 16 Infinity io-inert Quaternary L, Features, Technical etails, pplications, and Specifications, gilent Technologies ata Sheet, publication number EN, 1. The gilent 16 Infinity II WR with a io-inert standard flow cell is recommended for all common applications. It is especially well suited for bio analytical applications, for example, protein analysis using ion exchange chromatography (IEX) or size exclusion/gel filtration (SE). Low pressure applications around 1 bar, commonly used in protein analysis and analytical scale preparative L, showed high RT precision. This information is subject to change without notice. gilent Technologies, Inc., 17 Published in the US, pril 1, EN