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Agilent 1260 Infinity MethodDevelopment Solution Automatic Scaling of Gradient Times and Flow Rates forDifferent Column Lengths and Diameters Using theAgilent ChemStation Method Scouting Wizard

AbstractThe Agilent 1260 Infinity Method Development Solution offers:

• Automated setup of method development sequences with all needed methods forcolumn scouting, variation of mobile phases, gradients, and temperatures usingthe Agilent ChemStation Method Scouting Wizard

• Possible installation of eight columns of different selectivity, length, and internaldiameter

• Installation of up to eight columns of different scaling of flow rate and gradienttimes according to the column internal diameter and length

Author

Angelika Gratzfeld-Huesgen

Agilent Technologies, Inc.

Waldbronn, Germany

Application Note

Drug Discovery

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Agilent ZORBAX Eclipse Plus C-18

Agilent ZORBAX SB C-8

Agilent ZORBAX Eclipse Phenyl-Hexyl

Agilent ZORBAX SB CN

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IntroductionAnalytical HPLC method development isimportant in the chemical and pharma-ceutical industries. Typically, a set ofcolumns is tested in combination with aset of mobile phases. The scope of themethod scouting process is to find thebest column and mobile phase combi-nation for the separation of a specificset of compounds. After the columnscouting runs are finished fine-tuning ofthe gradient, column temperature, pH,and speed of the complete analysis isperformed with the Method ScoutingWizard. The entire, time-consumingprocedure can be shortened if columnsand solvents can be changed automati-cally during a sequence.

The Agilent 1260 Infinity MethodDevelopment Solution offers a highlyflexible system for one through eightcolumns up to 100 mm in length, or onethrough six columns up to 300 mm inlength. Several independent heatedzones are available to optimize the tem-perature for different columns. In addi-tion, solvent selection valves can beclustered with the pump to enhance theselection of available mobile phases.

In combination with the AgilentChemStation Method Scouting Wizard,sequences with different methods canbe created automatically, wherecolumns and solvents become methodparameters. This allows automated,unattended change of columns and solvents within a sequence.

This Application Note demonstrates:

• The use of the Agilent 1260 InfinityMethod Development Solution for col-umn scouting to find optimum separa-tion conditions for a specific applica-tion problem.

Results and discussionMethod development usingcolumns of different selectivity

In this study, we used the compoundsshown in Figure 1, which contain acidic,basic and neutral compounds.

A water/acetonitrile or water/methanol gradient was used and 5% ofa TFA solution (2 % TFA in water) wasadded over the complete run time tokeep the ionic strength constant. The Achannel contained the aqueous mobilephase, the B channel contained ace-tonitrile, the C channel methanol andthe D channel the 2% TFA solution.

Four columns of different selectivity,but the same length, internal diameterand particle size were used for methodscouting to find appropriate chromato-graphic conditions. The MethodScouting Wizard was used to create amethod scouting sequence.

A basic method must be set up to useas a starting point for the MethodScouting Wizard. This method is thenused to create other methods neededfor the different columns. In this exam-ple, the same flow rate and the same

• The use of the Method ScoutingWizard to scale flow rate, run timesand gradient times, if columns of dif-ferent internal diameter and length areused in the same sequence.

EquipmentAn Agilent 1260 Infinity Method

Development Solution was usedincluding:

• Agilent 1260 Infinity Quaternary Pump(G1311B)

• Agilent 1260 Infinity Autosampler(G1367E)

• Two Agilent 1260 InfinityThermostatted Column Compartments(G1316C)

• Agilent 1260 Infinity Diode ArrayDetector (G4212B)

• Agilent Method Development ValveKit, high pressure (G4230B) withAgilent Method Development CapillaryKit low dispersion, for short columns(p/n 5067-1595)

• Several Agilent ZORBAX columns ofdifferent lengths, internal diametersand chemistries

• Agilent ChemStation B04.02 withAgilent ChemStation Method ScoutingWizard add-on

Parabenes Toluene Phenol N,N-diethyl-m-Toluamide

Figure 1Chemical structure of first application example.

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gradient were applied for all columns.The resulting method developmentsequence is shown in Figure 2.

The Method Scounting Wizard leadsthrough 10 steps to set up the completesequence. The ternary gradient isdefined in Step 4 (Figure 3).

Figure 2Sequence created by the Method Scouting Wizard; column wash and equilibration runs can be included.

Figure 3Method Scouting Wizard screen for setting up ternary gradients.

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The sequence was completed afterapproximately seven hours. All fourcolumns were tested with two organicsolvents and two replicates for eachnew chromatographic condition. Afterthe sequence was finished the chro-matograms were overlaid and evaluated(Figure 4). Using acetonitrile as the

acetonitrile as the organic solvent. Thebest resolution was obtained using theAgilent ZORBAX SB CN column withmethanol as the organic solvent (Figure5). Even the peaks that showed coelu-tion on the CN phase using acetonitrileas the mobile phase could be separatedusing methanol.

organic phase the Agilent ZORBAXEclipse Plus C18, 100 × 4.6 mm, 1.8 µmcolumn showed the best resolution forall peaks.

Methanol was used as the secondorganic phase and chromatogramswere compared with those obtainedfrom the previous experiments using

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Agilent ZORBAX SB C-8

Agilent ZORBAX Eclipse Phenyl-Hexyl

Agilent ZORBAX SB CN

Figure 4Chromatograms of four columns using acetonitrile as the organic phase.

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Mobile phase: Water/ACN/TFA

Mobile phase: Water/MeOH/TFA

Figure 5Comparison of chromatograms with using either acetonitrile or methanol as the organic solvent on theAgilent ZORBAX Eclipse Plus SB-CN column. The upper chromatogram delivers the best resolution forall peaks.

Chromatographic conditions

Columns 4.6 × 100 mm, Agilent ZORBAX Eclipse Plus C-18 or SB C-8 or Phenyl-Hexyl or SB CN, 1.8 µm

Mobil phase: A = WaterB = AcetonitrileC = MethanolD = 2% TFA in water

Gradient ternary: 0 min 80% A, 15% B or C, 5% D

8 min 5% A, 90% B or C, 5% D

Flow rate: 1.5 mL/min

Stop time: 15 min

Post time: 5 min

Inj vol: 3 µL

Column temp: 40 °C

DAD: 254, 270, 220, 230/10 nm, Ref 360/100 nm,

Flow cell: 10 mm

Peakwidth: > 0.013 min (20 Hz)


Mobil Phase: A = WaterB = AcetonitrileC = MethanolD= 2% TFA in water

Gradient ternary: 0 min 80% A, 15% B or C, 5% D

8 min 5% A, 90% B or C, 5% D


Stop time: 15 min

Post time: 5 min

Inj vol: 3 µL

Column temp: 40 °C

DAD: 254, 270, 220, 230/10 nm, Ref 360/100 nm,

Flow cell: 10 mm

Peak width: > 0.013 min (20 Hz)

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In this example, two sets of chromato-graphic condition resulted in sufficientresolution for the nine peaks. One wasbased on a C18 phase using acetonitrileand the other solution was based on aCN phase and methanol (Figure 6).

Method development usingcolumns of different lengthand internal diameterWhen using columns with differentlengths or internal diameters within thesame method development sequence,the gradient times and flow rates mustbe adjusted to achieve comparableresults. The Method Scouting Wizardoffers an automated tool to scale bothparameters as shown in Figure 7.

Scaling of gradient and run timeGood separation was achieved by theAgilent ZORBAX Eclipse Plus C18, 4.6 ×100 mm column with acetonitrile asorganic phase (Figure 6). The length ofthe C18 phase used was varied from 50mm up to 150 mm to discover whichcolumn length is the most appropriatefor the application discussed. The inter-nal diameter for all columns remainedat 4.6 mm.

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Agilent ZORBAX Eclipse Plus C-18 ACNat max 356 bar

Agilent ZORBAX SB CN with MeOH max 535 bar

Uracil

Phenol

Methyl paraben

Ethyl paraben

Propyl paraben

N,n-diethyl-m-mToluamide

Heptyl parabenTolueneButyl paraben

Figure 6Two solutions for the separation of the nine peaks.

Figure 7Scaling of flow and gradient (run times) in the Method Scouting Wizard.

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The basic method was set up for thelongest column. The flow rate was setto 1.5 mL/min and the gradient time to8 min. The gradient times for the 100,75 and 50 mm column were automati-cally scaled to 5.34, 4.02 and 2.69 minby the Method Scouting Wizard (Figure 8).


Basic method Mobile phase: A = Water

B = Acetonitrile D = 2% TFA in water

Basic gradient for 150 mm length:

0 min 80 % A, 15 % B; 5 % D

8 min 5 % A, 90 % B, 5 % D

Gradient for 100 mm length:

0 min 80 % A, 15 % B; 5 % D

5.34 min 5 % A, 90 % B, 5 % D


0 min 80 % A, 15 % B; 5 % D

4.02 min 5 % A, 90 % B, 5 % D


0 min 80 % A, 15 % B; 5 % D

2.69 min 5 % A, 90 % B, 5 % D


Stop time: 15 min

Post time: 5 min

Inj vol: 3 µL

Column temp: 40 °C

DAD 254, 270, 220, 230/10 nm, Ref 360/100 nm,

Flow cell: 10 mm

Peak width: > 0.013 min (20 Hz)

The results for the different columnsregarding resolution and solvent con-sumption are compared in Table 1.

Column length Run time Gradient time Resolution marked Solvent consumption(mm) (min) (min) Peak /run (mL)

50 5 2.69 2.15 7.5

75 7.5 4.02 3.07 11.25

100 9 5.34 2.65 13.5

150 15 8 4.22 22.5(basic method)

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length = 100 mm, Tg = 5.34 min



Agilent ZORBAX Eclipse Plus C-18 with an id of 4.6 mmTg = gradient time

Figure 8Scaling of run time and gradient time according to column length by the Method Scouting Wizard (Tg= gradient time).

Table 1Results of column scouting with different column length. Flow rate was 1.5 mL/min for all columns.

The chromatograms show all the samepatterns. The best resolution wasobtained on the 150 mm column, butwith a 15 min run time leading to highersolvent consumption. A resolution > 2was obtained on the 50 mm columnwithin 5 min, which is typically suffi-cient for a separation. The 75 mmlength column provided the best com-promise between run time (7.5 min) andresolution (3.07).

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Scaling of flow rateIn a third experiment the internal diam-eter of the columns was varied becausecolumns from different vendors are notnecessarily available in the samedimensions. For example, the innerdiameter of the 4 mm columns can be 4 mm or 4.6 mm depending on the man-ufacturer. It is necessary to adjust theflow rate according to the diameter toget comparable results for thesecolumns. We selected two columnswith the same length and same particlesize but with internal diameters of 4.6and 3 mm to demonstrate flow ratescaling. The chromatograms are shownin Figure 9.

The flow rates were set automaticallyaccording to the internal diameter ofthe column used. For example, the flowrate of the 3 mm column was reducedto 0.638 mL/min. Typically slightlylonger retention times for columns withsmaller internal diameters are observed(Figure 9). This is because the flow ratescaling calculation is based on columnvolume and does not take the systemdelay volume into account. Anotheradvantage of the flow scaling option isminimal risk of errors due to over-pres-sure, because a column of smallerinternal diameter is switched into theflow path. Otherwise the completesequence would be stopped and aborted.

ConclusionThe Agilent 1260 Infinity MethodDevelopment Solution in combinationwith the Method Scouting Wizardallows automated column, gradient,mobile phase and temperature scoutingwith up to eight columns with differentselectivity, different length and internaldiameter. The user is guided by theMethod Scouting Wizard through allsetup screens. The sequence, methodsand required rinse and re-equilibrationsteps are created automatically once allthe necessary information is completed.In addition, the software adapts flowrate, gradient time and run time accord-ing to the used column dimensionsspecified. This optimizes the method forresolution or shortest run time in onesequence. Finally, this software mini-mizes the risk that the maximumallowed pressure for a column isexceeded or the sequence will bestopped due to an error.

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ID = 4.6 mmFlow rate = 1.5 mL/min

ID = 3.0 mmFlow rate = 0.638 mL/min

Columns with same length and particle size a length of 100 mm

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Figure 9Automatic scaling of flow rate for different column IDs by the Method Scouting Wizard.

Columns: Agilent ZORBAX SB-C8 3.0 × 100 mm, 1.8 µm (upper trace), Agilent ZORBAX Eclipse Plus C18 4.6 × 100 mm, 1.8 µm (lower trace)

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