Performance Evaluation Of The Agilent 1290 Infinity 2D-LC .

Performance evaluation of theAgilent 1290 Infinity 2D-LC Solutionfor comprehensive two-dimensionalliquid chromatographyTechnical Overview2D-LCConventional 1D-LCAbstractThis Technical Overview presents an example of a comprehensive setup fortwo‑dimensional liquid chromatography (2D-LC) based on the Agilent 1290 Infinity2D-LC Solution. The instrument configuration and the setup of the interfacebetween the first and second chromatographic dimension is described in detail.The analysis of a standard sample with statistical evaluation of the results isshown to demonstrate the performance of the instrument.

IntroductionThe separation of complex samples canbe improved by deploying a comprehen‑sive two-dimensional liquid chroma‑tography (LCxLC) system. In an LCxLCsystem, typically two pumps and twocolumns are used and fractions fromthe separation on the first column (firstdimension) are continuously trans‑ferred to a second column (seconddimension). This transfer is done byfilling two loops alternately controlledby valve switching.The advantage of LCxLC separationcompared to a standard LC separationis the increased peak capacity dueto the multiplicative behavior of thepeak capacities of the first and seconddimension1. This is an idealistic modelthat is only valid if completely orthogo‑nal separation mechanisms are usedfor the separation on the columns inthe first and second dimension2. Thisstate can be approximated for separa‑tions such as ion exchange in the firstand reversed phase separation in thesecond dimension. In reality, similarselectivities like different reversedphase selectivities are used. In thiscase, the peak capacity is decreasedand can be explained as an accessibletriangular area of the two-dimensionalchromatogram used for the separation3.This can be optimized by designingcomplex gradients for the separation inthe second dimension to increase theaccessible area used for separation andherewith increasing peak capacity.To achieve best separation perfor‑mance, a complex gradient wasdesigned for the second dimensionby a specialized software tool.SoftwareExperimental LCxLC Software for 2D-LC dataanalysis from GC Image LLC, Lincoln,NE, USAEquipmentThe Agilent 1290 Infinity 2D-LCSolution used for the performanceevaluation comprised the followingmodules: Two Agilent 1290 Infinity Pumps(G4220A) Agilent 1290 Infinity Autosampler(G4226A) with thermostat(G1330) Agilent 1290 Infinity ThermostattedColumn Compartment (G1316B)with 2-position/4-port duo valve(G4236A) for 2D-LC Agilent 1290 Infinity Diode ArrayDetector (G4212A) with 60-mmAgilent Max-Light flow cell(G4212-60007)In this configuration, the first andsecond dimension pumps are identical.Typically, the second dimension pumpmust be an Agilent 1290 Infinity pumpto deliver fast gradients to the seconddimension column. The first dimensionpump is flexible and could also be anAgilent 1260/1290 Infinity QuaternaryPump, an Agilent 1260 Infinity BinaryPump or an Agilent 1260 InfinityCapillary Pump.2 Agilent OpenLAB CDS ChemStationEdition, version C.01.03 with 2D-LCadd-on softwareColumnsFirstdimension: Agilent ZORBAX RRHDEclipse Plus C18,150 2.1 mm, 1.8 µmSeconddimension: Agilent ZORBAX RRHDEclipse Plus Phenyl Hexyl,50 3.0 mm, 1.8 µmMethodFirst dimension pumpSolvent A: Acetonitrile 0.1% formic acidSolvent B:Water 0.1% formic acidFlow rate:0.1 mL/minGradient:5% B at 0 min95% B at 30 min95% B at 40 minStop time:40 minPost time:15 min

Second dimension pumpSolvent A: Methanol 0.1% formic acidSolvent B:Water 0.1% formic acidFlow rate:3 mL/minInitialgradient:5% B at 0 min15% B at 0.5 min5% B at 0.51 min5% B at 0.65 minFigure 12D-LC Gradient profile.1st dimension Gradient (red): 0 min 5% B–30 min 95% B, 40 min–95% B. Stop time: 40 min.Post time: 15 min. 2nd dimension Gradient (blue): Initial Gradient: 0 min–5% B, 0.5 min–15% B, 0.51 min–5% B,0.65 min–5% B. Gradient Modulation: 0 min 5% B to 30 min 50% B, 0.5 min 15% B to 30 min 95% B, 0.51 min 5% to30 min 50% B, 0.65 min 5% B to 30 min 50% B.Gradientmodulation: 5% B at 0 min to50% B at 30 min15% B at 0.5 min to95% B at 30 min5% B at 0.51 min to50% B at 30 min5% B at 0.65 min to50% B at 30 minThe final second dimension gradient isshown in Figure 1. The user interfacefor setup of the second dimensiongradient is shown and explained inFigure 2.Figure 2Window for the set-up of the second dimension gradient in a 2D-LC separation.A) Nested table for the set-up of the 2D-LC gradient and gradient shift.B) Table for the set-up of time, and peak-based triggering events to start 2D-LC separation.C) Graphic display of the 2D-LC gradient according to the set-up in the 2D-LC gradient table A and B. The gradientcan be changed by drag and drop of the points of the curve in this graphical interface. The changed values arewritten back to the gradient table.D) Graphical display of a single gradient snip in the second dimension which is repeated by the modulation rate andshifted as given in the gradient table.E) Calculations which help to set-up the 2D-LC separation experiment from currently used LC parameters.3

Thermostatted columncompartment First dimension column at left sideat 25 C. Second dimension column at rightside at 60 C. Two 80 µL loops are connected tothe 2-position/4-port duo valve andare located at the left side. The valveis switched automatically after eachsecond dimension modulation cycle.The complete plumbing is shown inFigure 3. In this case, the loops areused in a countercurrent manner(the loops are filled and eluted fromdifferent sides).AutosamplerLoop tor2D-ColumnLoop 2Loop ump2D-Column Injection volume: 5 µL Sample temperature: 8 C Needle wash: 6 s in methanolLoop 2Figure 32D-LC with 2-position/4-port duo valve interface configuration, green arrow (&) Fill-direction,red arrow (&) Analyze-direction, (LCxLC, countercurrent).Diode array detector Wavelength: 260/4 nm,Ref. 360/16 nm Slit: 4 nm Data rate: 80 Hz Flow cell: 60 mm Max-Lightflow cellChemicalsAll solvents used were LC grade.Acetonitrile and methanol were pur‑chased from Merck, Germany. Freshultrapure water was obtained from aMilli-Q Integral system equipped witha 0.22 μm membrane point-of-usecartridge (Millipak).RRLC Checkout Sample containing ace‑tophenone, propiophenone, butyrophe‑none, valerophenone, hexanophenone,heptanophenone, octanophenone,benzophenone and acetanilide waspurchased from Agilent Technologies(p/n 5188-6529). Gradient EvaluationMix containing uracil, phenol, methylparaben, ethyl paraben, propyl paraben,butyl paraben, and heptyl parabenwas purchased from Sigma-Aldrich(catalog no. 48271). Reversed PhaseTest Mix containing uracil, phenol,N,N-Dimethyl-m-toluamide and toluenewas purchased from Sigma-Aldrich(catalog no. 47641-U). Sulfamethazine(Stock solution: 100 µg/mL,catalog no. S6256) and 2-hydroxyquinoline (Stock solution: 100 µg/mL;catalog no. 270873) were purchasedfrom Sigma-Aldrich.4SampleThe standards were mixed to a 2D-LCtest sample as following: RRLC Checkout Sample: 100 µL Gradient Evaluation Mix: 800 µL Reversed Phase Test Mix: 200 µL Sulfamethazine: 100 µL 2-hydroxy quinoline: 100 µL

Results and discussionThe maximum peak capacity in atwo‑dimensional LC separation canonly be achieved in cases where theseparation mechanisms of the twodimensions are truly orthogonal. Ifthey are similar, only a triangular rangeof the separation area is used. Thisbecomes especially important whentwo reversed phase separations arecombined in a 2D-LC experiment suchas separation on a C18 phase for stand‑ard separation and a hexylphenyl phasefor selective separation of aromaticcompounds. Typically, the separatedcompounds are narrowly distributedaround a diagonal line in the separationdiagram (Figure 4.1).In the optimized separation, allcompounds were clearly separatedin the second dimension, even com‑pounds co-eluting from the firstdimension (Figure 5).Figure 4Optimization of the 2nd dimension gradient. Red line in bottom pictures shows the 1st dimension gradient and the blueline the repeating 2nd dimension gradient.1) 2nd dimension gradient repeats between 5% and 95% organic2) First adjustment of the second dimension gradient to improve separation by lower organic in the seconddimension for the compounds of higher polarity3) Second adjustment to the same maximum organic composition as given from the first dimension at each top ofthe 2nd dimension gradient4) Increase in organic starting composition during the runtime to improve separation and focus the compounds bybetter usage of separation time in the 2nd dimension 103 103AColumn I location 21.450BColumn I location 21.45015Intensity30IntensityThis is especially true when a simplegradient is used for the seconddimension. The separation in thesecond dimension can be optimizedfor an improvement in separation by amodification of the second dimensiongradient. This is done with the spe‑cial 2D-LC software tool for gradientsetup. In the first step (Figures 4.2and 4.3) the second dimension gradi‑ent maximum is adapted to the firstdimension maximum at the respec‑tive time. This opens the separationangle and improves separation, but thecompounds lose their focus and showdispersion along the separation. Toavoid this and to move them into themiddle of the separation, the startingcomposition of the gradient is alsomoved to a higher organic level duringthe separation in the second dimension(Figure 4.4) which achieves optimumfocusing, area and separation.201051000051015 20 25Column II3035051015 20 25Column II3035Figure 5Comparison of peaks eluting at 21.45 minutes from the first dimension separated acceding to the second dimensiongradient shown in Figure 3.1 (A) and 3.4 (B)A) Peaks of compound 9 and 10 eluting at 27.78 sec and 29.51 secB) Peaks of compound 9 and 10 eluting at 22.65 sec and 26.30 sec5

The compounds could be detected bythe 2D-LC data analysis software andare annotated as red circles (Figure 6).The retention times in the first andsecond dimension are given inTable 1. The compound number 1 isuracil, which shows the dead timeof 4.55 minutes for the first dimen‑sion and 8.32 seconds for the seconddimension. Compound number 20 isoctanophenone eluting at the end ofthe separation at 33.80 minutes/27.94 seconds.Figure 6Detection of all used compounds in the optimized 2D-LC separation. The compounds are separated in the seconddimension, even compounds which are co-eluting from the first dimension in the same time segment are separatedin the second dimension. The detected compounds are annotated by round red circles. The numbers correspond to thefollowing list:1) uracil, 2) sulfamethazine, 3) 2-hydroxy quinoline, 4) acetanilide, 5) phenol, 6) methyl paraben, 7) acetophenone,8) ethyl paraben, 9) propyl paraben, 10) N,N-Dimethyl-m-toluamide, 11) propiophenone, 12) butyl paraben,13) butyrophenone, 14) toluene, 15) benzophenone, 16) valerophenone, 17) hexanophenone, 18) heptyl paraben,19) heptanophenone, 20) octanophenoneCompoundRT I (min)RT II droxy .6014.566Methyl paraben16.2520.957Acetophenone18.8520.588Ethyl paraben18.8522.889Propyl 3811Propiophenone22.7519.8112Butyl 27.9521.2817Hexanophenone29.9022.9518Heptyl ophenone33.8027.94Table 1Compounds in the 2D-LC test mixture.RT I (min): Retention time in the first dimension in minutesRT II (sec): Retention time in the second dimension in seconds6