teledyneisco.comTime-On-Target: Easy Method Development for Reverse Phase Preparative ChromatographySilver, J.E., Teledyne ISCO, Lincoln, NE, United States

AbstractA technique was developed to easily create optimized preparative HPLC and flash chromatography reverse phase methods from analytical HPLC/UHPLC runs. Preparative

chromatography methods for normal phase chromatography are easily created from thin-layer chromatography (TLC) plates. Reverse phase methods are more difficult to create

because TLC plates require a significantly longer time to run. Using HPLC/UHPLC for method development often requires complex scale-up calculations to determine gradient

segment lengths to effect the same resolution.

Time-on-Target uses a model compound to set a desired retention time on the preparative HPLC or flash chromatography system. The determined solvent composition from the

preparative system is then used to calibrate the scouting gradient used on the analytical LC system. Compounds to be purified are run using the same scouting gradient as that used for

the initial analytical calibration. Their retention time is adjusted by the calibrated scouting gradient to calculate a solvent composition which centers on an efficient focused gradient.

The determined gradient is fast, saving solvent and reducing waste. Once calibrated, reverse-phase method development for reverse phase chromatography is faster than that using

TLC for normal phase.

BackgroundCreation of a useful preparative gradient which offers high resolution and fast purification for natural products was difficult and time consuming. Most chemists run a “scouting gradient”

as a matter of course which provides useful information about compound purity and suggests the solvent composition to purify the desired compound(s). Due to various lags in the

gradient made by the pumping system, the actual preparative elution composition could only be guessed at.

One method to generate focused gradients is to measure the dwell and column volume of the analytical system to determine the gradient delay. The dwell volume is the volume from

the mixer to the head of the column, including sample loops. The column volume is the portion of the column filled with mobile phase. The gradient delay is subtracted from the target

compound’s elution time to determine the actual B solvent concentration. We determined that an additional factor, which we termed a “mixing volume”1, was required to generate a

more accurate B solvent concentration. This mixing volume was analogous to the “correlation factor” described by Gonnet2, the “instrument constant” mentioned by Blom3, and also

described as a “mixing volume” by Magee4. These techniques didn’t easily convert to flash chromatography and were, therefore, of limited utility.

Calibration of Prep System and Flash SystemsThis requires three steps and is done in a similar fashion for a preparative HPLC or a Flash Chromatography system. This step allows compensation for the gradient delay in the

preparative system.

1. Determine the dwell volume. This only needs to be done once. Replace one solvent with a solvent that absorbs UV light. Acetone is useful since it is miscible with water and easily

washed from the system. Replace the column with a union. Run isocratically with no absorbing solvent, then program a step gradient. Note the delay in absorbance from the step

and multiply be the flow rate to obtain the dwell volume. This was determined to be 14 or 18 mL for an CombiFlash NextGen 300+ (depending on flow rate) and 7.0 mL for

a ACCQPrep HP150 with a 5 mL loop.

2. Determine the column volume. This needs done once per column size and chemistry used; however, replacement columns of the same type will have the same column volume.

For example, all RediSep 20x150 C18 columns will have the same column volume, but this may be different from a RediSep 20x150 mm C18Aq column. Run the column with 10%

organic, and inject a small amount of sodium iodide or sodium nitrate while monitoring 215 nm. Note the peak elution time and multiply by the flow rate to obtain the column

volume. This step, and the one above, allow the calculated gradient to be adjusted for the delays of the preparative system. This step is not needed for RediSep Flash columns

because the column volume is printed on the label.

3. Set the elution time for the model compound using an isocratic run. Use the same solvent and modifiers as typically run on the analytical system. Adjust the mobile phase

composition to elute the compound at the desired time for the preparative runs, using the flow rate appropriate for the column. Model compounds used include ethyl paraben,

phenacetin, and N-benzylbenzamide. These were chosen because they elute at ~50% organic solvent for the C18 columns used. This step sets the retention time for the column used.

Calibration of phenacetin in methanol on a 15.5 g RediSep Gold C18 column (PN 69-2203-334 left) and a 20 x 150 mm RediSep Prep C18 column (PN 69-2203-826)on an ACCQPrep HP150 (PN 68-5230-053, right). Note that phenacetin gives a desired retention at 50% B in both cases.

Calibration of Analytical SystemThis only requires running the model compound with the scouting gradient used to evaluate synthesized compounds. This gradient typically starts at 5 or 10% and runs to 100% organic with no isocratic hold at the start. The same solvent system used to calibrate the preparative system, including modifiers, is used for this step. Columns with matching chemistries should be used.

After calibration, the programmed gradient time for the calibration compound may be calculated using the equations to the left.

Ma = Analytical gradient slope. P = programmed gradient %B; %BI = %B eluting compound in prep isocratic run; %BSa = Starting %B for analytical gradient; %BEa = End %B for analytical gradient; La = Gradient length for the analytical run. The gradient delay (Da) is the difference between the actual elution time and the programmed gradient (P) for the calibration compound. The concentration of strong solvent used to elute the compound is %B.

Sample from prep calibration run on an Agilent HPLC using an 4.6x150 mm RediSep Prep C18 column (PN 69-2203-800) with a water/methanol gradient. The peak eluting at 6.530 minutes is deemed to elute at 50% methanol.

For this example:

Ma = (100-5)/6 = 15.83

P = (50-5)/15.83 = 2.842 minutes

The Gradient Delay (Da) = 6.53-2.842 = 3.6879 minutes

Calculation of Focused GradientThis requires four steps:

1. Run the compound to be purified on the analytical system using the same gradient as the initial calibration.

2. Using the calculated value Da, determine the actual %B which elutes the compound.

3. Set a focused gradient encompassing the calculated %B.

4. Correct the gradient for the dwell and column volumes of the preparative system previously determined.

BYTeledyne ISCO

1 Silver, J.E.; Lewis; R.L, Fowler, N.; Letteney, E.N. Preparative Method Development from Analytical Columns. Presented at the 253rd American Chemical Society National Meeting, San Francisco, USA. Poster MEDI 487.2 Koza, P.; Gonnot, V.; Pelleter, J. Right-First-Time Isocratic Preparative Liquid Chromatography-Mass Spectrometry Purification. ACS Comb. Sci. 2012, 14, 273−279.3 Blom,K.F.; Glass, B.; Sparks, R.; Combs, A.P. Preparative LC-MS Purification: Improved Compound-Specific Method Optimization. J. Comb. Chem. 2004, 6, 874-883.4 Magee, M. H.; Manulik, J. C.; Barnes, B. B.; Abate-Pella, D.; Hewitt, J. T.; Boswell, P. G. Journal of Chromatography A. 2014, 1369, 73–82.

The corrected solvent composition for the desired compound is %BCorr ; TEa = the elution time for the desired compound in the analytical run. The other terms are the same as the earlier equations.

Crude piperine was run on an Agilent UHPLC using a 4.6x150 mm RediSep Prep C18 column in water/methanol and eluted at 7.769 minutes. The retention is very different from the calibration compound and thus serves as a useful example.

For this example, %BCorr = (7.769-3.6879) *15.83+5 = 69.63 %B

Set a focused gradient with the desired range (R), centered around this number- usually a range of 10% to 20%.

The final steps to calculating the gradient involve correcting the focused gradient for the preparative system by increasing the strong solvent concentration. For the equations to the left, Dp, the preparative system delay is determined by adding the prep system dwell volume (VDp) to the prep column volume (VCp) and dividing by the prep flow rate. The amount to increase the strong solvent concentration (Δ%B) is calculated by dividing the range (R) by the prep gradient length (Lp) then multiplying by Dp.

For an NEXTGEN 300+, VDp = 18.0 mL VCp = 42.5 mL (RediSep Rf Gold C18, PN 69-2203-336), run at 60 mL/min. The chosen range (R) is 10%, with a length (Lp) of 12 column volumes.

Dp = (18+42.5)/60 = 1.009 min

Δ%B = 10/12*1.009 = 0.84%

The final gradient is 70.47% ±5, or 65 to 75% methanol

Crude (125 mg dissolved in 1 mL DMSO) run on a RediSep Rf Gold C18 column (PN 69-2203-336) column in water/methanol gradient using the calibration described in this poster eluted at the expected time.

A simplified version of the algorithm was applied for the preparative HPLC run. The parameters were programmed into the PeakTrak software so that a scouting run on the ACCQPrep HP150 (PN 68-5230-054) equipped with a PurIon L mass spectrometer (PN 68-5237-084) allowed calculation of the focused gradient. A water/acetonitrile gradient was used. 0.050 mg sample was loaded.

0 2 4 6 8 10 12 14 160.0

0.1

0.2

0.3

0.4

0.5

Time (minutes)

AU (2

54 n

m)

0102030405060708090100

%B

(MeO

H)

0 2 4 6 8 10 120

500

1000

1500

mAU

(250

nm

)

Time (minutes)

0

50

100

%B

(MeO

H)

6.530

7.029

BI = 50%Da =3.6879 min

%BSa= 5%

%BEa =100%

Cor

rect

ed %

B (M

eOH

)

2.842 min (P)

La

Ma= (%BEa-%BSa)

La

P = (%BI-%BSa) Ma

%BCorr= (TEa-Da * Ma+%BSa)

DP=(VDp+ VCp) Fp

Δ%B = R * Dp Lp

0 2 4 6 8 10 120

500

1000

1500

2000

2500

mAU

(250

nm

)

Time (minutes)

0102030405060708090100

%B

(MeO

H)

Cor

rect

ed %

B (M

eOH

)

%B Corr= 69.6% MeOH

%BSa= 5%

%BEa=100%

7.769(T

Ea )

0 2 4 6 8 10 12 14 16 180.0

0.5

1.0

1.5

2.0

2.5

AU (2

14, 2

54 n

m)

Time (Min)

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1.0x1010

1.5x1010

Ion

Cur

rent

(201

, 201

, 286

, 340

, 308

, 340

Da)

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N)

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0.0

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AU (2

14 ,

254

nm)

Time (CV)

0

1x1010

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4x1010

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6x1010

7x1010

8x1010

Ion

Cur

rent

(100

-200

0 D

a)0

50

100

% B

(AC

N)

0 2 4 6 8 10 12 14 160

1

AU (2

14, 2

54 n

m)

Time (CV)

0

2x109

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8x109

1x1010

Ion

Cur

rent

(286

Da)

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(MeO

H)