UHPLC Method Transfer Using Fused-Core Columns€¦ · Method Transfer Scenarios A and D: No Change...

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TECHNICAL REPORT UHPLC Method Transfer Using Fused-Core Columns

Transcript of UHPLC Method Transfer Using Fused-Core Columns€¦ · Method Transfer Scenarios A and D: No Change...

Page 1: UHPLC Method Transfer Using Fused-Core Columns€¦ · Method Transfer Scenarios A and D: No Change in HALO Column ID Isocratic Method When an isocratic method on a HALO column is

T E C H N I C A L R E P O R T

UHPLC Method Transfer Using Fused-Core

Columns

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Most of the methods developed on UPLC® and UHPLC systems with sub-2-μm totally porous particle columns utilize 2.1- and 3.0-mm ID columns, because those column IDs require lower volumetric flow rates to produce the higher linear velocities that such small particles require to achieve their maximum performance. It is much easier and more convenient to transfer a method developed on a HALO Fused-Core UHPLC column from a UPLC or UHPLC system to a conventional HPLC sys-tem, because the HALO column backpressure is only 40–50% of that generated by a comparable sub-2-μm column under the same conditions. In fact, a method developed on a sub-2-μm column typically generates too much backpressure to move the original UHPLC column to an HPLC instrument, which has a pressure maximum of only 400 bar (6000 psi). On the other hand, a UHPLC method developed on a HALO column can be moved directly to the HPLC system, or in some cases, one can simply use a larger diameter HALO column (e.g., 3.0 mm vice 2.1 mm ID, or 4.6 mm ID vice 3.0 mm ID) of the same length for the HPLC method. Of course, appropriate modifications still must be made to the HPLC system to allow the highest possible performance from the HALO UHPLC column.

In Table 1, general recommendations are presented that describe the method and instrument parameters that should be considered and adjusted to transfer a method from a UPLC/UHPLC to an HPLC using a HALO Fused-Core column. In addi-tion, several different method transfer scenarios using HALO columns are shown in

UHPLC Method Transfer Using HALO Fused-Core Columns

Introduction

Figure 1. These scenarios include transferring a method from UHPLC to HPLC: (1) directly from a 2.1 or 3.0 mm ID HALO column, or, (2) indirectly from a 2.1 or 3.0 mm ID column to a 3.0 or 4.6 mm ID HALO column. Isocratic methods are simpler to transfer, although they are much more dependent on minimizing extracolumn vol-ume and dispersion than gradient methods. Practically speaking, to obtain optimum performance for the HALO column on an HPLC system, the extracolumn volume and extracolumn dispersion of the HPLC system must be minimized. Recommenda-tions for optimizing the performance of an HPLC system have been described earlier in a previous technical note (Reference 2), but, for convenience, are summarized in Table 1 for the scenarios shown in Figure 1.

In this technical report we discuss how to make method transfer easier from UHPLC to HPLC using HALO Fused-Core columns. We begin by summarizing recom-mendations for successful method development and transfer, including how to choose an appropriate column length and flow rate, as well as a general strategy for method development and transfer. We then provide estimates of maximum useable flow rates with both acetonitrile/water and methanol/water mobile phases for column tempera-tures from 25-60° C. The remainder of the technical note discusses some isocratic and gradient examples in detail, and also presents the results of a real gradient method transfer example using several HALO geometries

1 | UHPLC METHOD TRANSFER USING HALO FUSED-CORE COLUMNS

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Recommendations for Developing UHPLC Methods that are Transferrable to HPLC using HALO Fused-Core Columns

We have summarized some recommended steps for developing a UHPLC method, capable of being transferred to an HPLC, using HALO columns below.

1. Choose the column ID, length, and stationary phase for the method. a. Use 20, 30 and 50 mm lengths for less demanding separations b. Choose 75, 100, and 150 mm lengths for more difficult separations such as related substances methods, complex samples, impurity profiling, etc.

2. Set the flow rate for the chosen column ID. (see below)

3. Adjust the flow rate and column temperature so that the worst case UHPLC method backpressure is ≤ 80% of the HPLC instrument maximum backpressure (400 bar) or whatever your target maximum desired back pressure. Refer to Table 1 for approximate maximum flow rates for different column geometries and at various temperatures using either acetonitrile or methanol as organic modifier (see Note below).

4. Optimize the isocratic or gradient separation using the most effective parameters for adjusting RPLC selectivity. a. Column stationary phase (C18, C8, RP-Amide, Phenyl-Hexyl, PFP, ES-CN) b. Organic modifier choice (ACN, MeOH, ACN/MeOH blend) c. % Organic modifier (isocratic) or gradient steepness d. Mobile phase pH (for ionizable analytes) e. Column temperature

5. Follow the guidance given in Table 2 and Reference 2 for minimizing the extra column volume and dispersion of the HPLC system.

6. Transfer the UHPLC method to the HPLC system, using the same or larger diameter HALO column, and make the adjustments to the injection volume, flow rate, and gradient program, and injection delay as described later.

7. Assess the performance of the transferred method on the HPLC system.

To obtain superior performance from a HALO column on a UHPLC or HPLC system, a suitable combination of flow rate and column temperature is needed. Note that optimum linear velocity increases as column temperature increases, but the corresponding efficiency (or peak capacity for gradient separations) tends to stay approximately constant. Typically, higher efficiencies and peak capacities can be obtained using linear velocities of approximately 2.5 mm/sec or higher. These velocities correspond to the following flow rate ranges for the respective HALO column IDs:

2.1 mm: ≥ 0.25 mL/min 3.0 mm: ≥ 0.5 mL/min 4.6 mm: ≥ 1.25 mL/min

However, to develop a method on a UPLC or UHPLC system that must be trans-ferred to an HPLC system, it is important to choose a combination of flow rate, sol-vent composition, and column temperature that will not exceed (for the most viscous mobile phase composition) 80% of the maximum backpressure of the HPLC system (typically 400 bar). Use the information provided in Table 1 for the maximum useable flow rates predicted for various HALO column geometries with acetonitrile/water and methanol/water mobile phase mixtures for temperatures between 25 and 60°C. For other temperatures, you can interpolate the values for the maximum flow rates for a given geometry. The worst case backpressures for acetonitrile/water and methanol/water mixtures occur between 10-20% and 40-50% organic, respectively.

Note: The backpressure for a UHPLC system will typically be higher, at the same flow rate, than the backpressure on an HPLC system, because of narrower ID tubing used with UHPLC systems.

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Table 1: Estimating the Maximum Acceptable Flow Rate for Methods Developed on a UHPLC System that will be transferred to an HPLC system using HALO Columns

Mobile Phase: Acetonitrile and WaterHALO Column ID: 2.1 mmMaximum Acceptable Flow Rate (mL/min)

Mobile Phase: Methanol and WaterHALO Column ID: 2.1 mmMaximum Acceptable Flow Rate (mL/min)

Mobile Phase: Acetonitrile and WaterHALO Column ID: 3.0 mmMaximum Acceptable Flow Rate (mL/min)

Mobile Phase: Methanol and WaterHALO Column ID: 3.0 mmMaximum Acceptable Flow Rate (mL/min)

To estimate the maximum flow rates, it was assumed that ~60 cm of 0.005” ID tubing was used in the sample flow path. Viscosities for acetonitrile/water and methanol/ water mixtures as a function of temperature were taken from an online calculator (lcsyscal.exe) and are comparable to those available in Introduction to Modern Liquid Chromatography, L.R. Snyder, J.J. Kirkland, J.W. Dolan, Willey, 2010, 3rd edition, p 884. The flow resistance parameter for HALO columns was set at 653. Estimated flow rates do not include pressure drops from autosampler and flow cell.

20 1.6 1.8 2.2 2.7 3.2

30 1.1 1.3 1.5 1.7 2.2

50 0.7 0.8 1.0 1.2 1.4

75 0.5 0.5 0.6 0.8 0.9

100 0.4 0.4 0.5 0.6 0.7

150 0.2 0.3 0.3 0.4 0.5

20 3.0 3.3 4.0 4.9 5.0

30 2.2 2.4 2.9 3.5 4.2

50 1.4 1.5 1.9 2.2 2.7

75 0.9 1.1 1.3 1.5 1.8

100 0.7 0.8 1.0 1.2 1.4

150 0.5 0.5 0.7 0.8 1.0

20 1.0 1.1 1.4 1.7 2.0

30 0.7 0.8 0.9 1.2 1.4

50 0.4 0.5 0.6 0.7 0.9

75 0.3 0.3 0.4 0.5 0.6

100 0.2 0.2 0.3 0.4 0.4

150 0.1 0.2 0.2 0.2 0.3

20 1.9 2.1 2.5 3.0 3.1

30 1.3 1.5 1.8 2.2 2.6

50 0.9 0.9 1.1 1.4 1.7

75 0.6 0.7 0.8 1.0 1.1

100 0.4 0.5 0.6 0.7 0.9

150 0.3 0.3 0.4 0.5 0.6

Column Length (mm)

Column Length (mm)

Column Length (mm)

Column Length (mm)

Column Temperature (°C) Column Temperature (°C) Column Temperature (°C) Column Temperature (°C)

25 30 40 50 60 25 30 40 50 60 25 30 40 50 60 25 30 40 50 60

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FIGURE 1: Various Scenarios for Method Transfer from UHPLC to HPLC Using HALO Columns with Different IDs

HALO 3.0 mm x LHALO 3.0 mm x L

HALO 4.6 mm x LHALO 3.0 mm x L

HALO 4.6 mm x LHALO 2.1 mm x L

HALO 3.0 mm x LHALO 2.1 mm x L

HALO 2.1 mm x LHALO 2.1 mm x L

TAbLE 2: Selection of Method and Instrument Parameters for Successful Method Transfer Using Various HALO Column IDs

FIGURE 2: Schematic Diagram of High Pressure Gradient System Showing Delay Volume

Column Internal Diameter (mm)

2.1 3.0 4.6

Mobile Phase A

Mobile Phase B

Degasser Column Heater

Heat Exchanger

Flow CellColumn

DetectorPump A

Pump B

Autosampler

Mixer

Waste

Delay Volume (dwell volume)includes all volume from where solvents are mixed up to column inlet

HIGH PREssURE MIxING GRADIENT sysTEM

Injection Volume (uL) 0.5 <_ Vinj <_ 5 1 <_ Vinj <_ 5 1 <_ Vinj <_ 10

Sample Solvent* %B <_ mobile phase %B <_ mobile phase %B <_ mobile phase

Tubing Length minimize minimize minimize

Tubing ID (mm) <_ 0.13 <_ 0.13 <_ 0.13

Tubing Length (mm) minimize minimize minimize

Tubing ID (mm) 0.075 <_ ID <_ 0.13 ID <_ 0.13 ID <_ 0.13

Flow Cell Volumn (uL) 0.5 <_ Vcell <_ 2 2 <_ Vcell <_ 5 2 <_ Vcell <_ 5

Data Rate (Hz) > 10 > 10 > 10

Time Constant (sec.) <_ 0.1 <_ 0.1 <_ 0.1

Heat Exchanger(s) (uL) < 2 < 2 <_ 3

Tubing, before column

Injector

Tubing, after column

Detector

Column Heater

A

B

C

D

E

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* Note: It is not always possible or practical to use a sample solvent that is weaker than the starting mobile phase. In those cases, keep the injection volume as small as possible

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Method Transfer Scenarios A and D: No Change in HALO Column ID

Isocratic Method

When an isocratic method on a HALO column is transferred from UHPLC to HPLC, the flow rate and injection volume can be the same as those used with the UHPLC system, not withstanding any possible differences in flow cell path length or design or in the responses of the different detectors. Recommendations are given in Table 2 for the adjustments necessary to minimize extracolumn dispersion. Gradient Method

When transferring a gradient method from UHPLC to HPLC using a HALO column, the same flow rate and injection volume can be used (Table 2), unless the UHPLC backpressure exceeds 320–350 bar. Additionally, to ensure comparable gradient separations, the effective gradient delay volume for the HPLC system must be adjusted to match that of the UHPLC system. This adjustment is usually most important for the earliest eluting components in the gradient, and, if not adjusted properly, can affect relative retention order and peak widths for those components. The gradient delay volume is the system volume from the point at which the A and B solvents are mixed to the inlet of the column itself (Figure 2). Normally, the delay volume of the UHPLC system will be much lower than that of the HPLC system. To adjust the effective delay volume of the HPLC system, an injection delay is used to allow the gradient program on the HPLC system to progress long enough to match the actual delay volume of the UHPLC system, before the sample is injected. Many UHPLC and HPLC systems allow a programmable injection delay or the use of an autosampler injector program to make such an adjustment. For example, if a UHPLC system has a gradient delay volume of 120 μL and the UHPLC method uses a flow rate of 0.5 mL/min for a 2.1 mm ID HALO column, then for an HPLC system with an 800 μL delay volume and the same 0.5 mL/min flow rate, one would set an injection delay of (800 – 120) μL/500 μL/min = 1.36 minutes.

Gradient Program and Equilibration Time

When transferring a UHPLC gradient method to an HPLC system with a larger delay volume, it’s also important to remember to include sufficient time for the mobile phase composition at the column to “catch up” to the gradient program on the HPLC system. This factor is sometimes forgotten, which can lead to confusion and poor repeatability for those transferring methods. See Example 1 below example 1

In Table 3, the gradient program for the UHPLC system is shown on the left and that for the HPLC system is shown on the right. The delay volume for the UHPLC system is 120 μL, and that for the HPLC system is 800 μL, as per the discussion above. The same HALO column (2.1 x 50 mm, 2.7 μm) and method are moved from the UHPLC to the HPLC system, and the same flow rate of 0.5 mL/min is used. The UHPLC and HPLC gradient programs are nearly identical, except that a longer hold time for the HPLC system is set to allow the column to equilibrate fully. For both instruments, 5 column volumes and 2 system volumes (gradient delay volumes) are used for column equilibration. Note the impact of the larger delay volume of the HPLC system (Figure 3). If the identical gradient program were to be used on the HPLC system, there would not be adequate time for column equilibration, because of the HPLC system’s much larger delay volume. The effective hold time at the starting %B (1.6 min at 5% B [column]) is much longer on the HPLC system due to its ~7-fold larger delay volume, and an additional 1.6 minutes of hold time (0.8 mL/0.5 mL/min) is also added to allow the column to “catch up” to the gradient program (Figure 3). Some types of instrument software allow you to set a “post time” at the end gradient method (instead of building the equilibration time into the actual gradient program); so that data collection can stop and disk space can be saved. If a “post time” is used, ensure that the sum of the post time and the method run time is long enough to allow sufficient or the desired amount of column equilibration.

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Table 3: Comparison of Gradient Programs and %B at Column for UHPLC System and HPLC System for Method Transfer using 2.1 × 50 mm HALO Column

FIgure 3: Comparison of Gradient Program and Actual Composition Program at Column Inlet for a UHPLC System with 100 µL Delay Volume and an HPLC System with 800 µL Delay Volume

Time %B Time %B (column)

0.00 5 0.00 5

5.00 55 0.20 5

6.00 55 5.20 55

6.10 5 6.20 55

7.37 5 6.30 5

Stop 8.00 7.57 5

Time %B Time %B (column)

0.00 5 0.00 5

5.00 55 1.60 5

6.00 55 6.60 55

6.10 5 7.60 55

10.17 5 7.70 5

Stop 12.00 11.77 5

Vinj 1.0 µL

VD 0.12 mL

ID 2.1 mm

Length 50 mm

VM 0.087 mL

F 0.5 mL/min

Linear Velocity (µ) 4.8 mm/sec

Vinj 1.0 µL

VD 0.800 mL

ID 2.1 mm

Length 50 mm

VM 0.087 mL

F 0.5 mL/min

Linear Velocity (µ) 4.8 mm/sec

uHplC SySTem HplC SySTem

Program

UHPLC Actual 100 µL Delay Volume

HPLC Actual 800 µL Delay Volume

60

50

40

30

20

10

00.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

Time (min.)

%B

(AC

N) a

t C

olum

n

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Note: These comparisons ignore the gradient rounding that can occur with many systems

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Method Transfer Scenarios B, C and E: Change to Larger HALO Column ID

Isocratic Method

In some situations, it is easier to transfer a UHPLC method developed on a 2.1- or 3.0-mm ID HALO column to a larger-ID column of the same length for use on an HPLC system. When transferring a UHPLC method to a larger-ID HALO column, it is necessary to scale the flow rate (linear velocity) and injection volume to compensate for the increased volume of the larger-ID column. Both the flow rate and injection volume should be increased by the square of the ratio of the respective column internal diameters as shown in the following equations:

F3.0 mm = F2.1 mm × (3.0 mm/2.1 mm)2

Vinj (3.0 mm) = Vinj (2.1mm) × (3.0 mm/2.1 mm)2

Analogous equations are applied for scaling flow rate and injection volume when moving from 3.0 mm to 4.6 mm ID columns.

F4.6 mm = F3.0 mm × (4.6 mm/3.0 mm)2

Vinj (4.6 mm) = Vinj (3.0 mm) × (4.6 mm/3.0 mm)2 Gradient Method

To transfer a gradient UHPLC method developed on a 2.1 or 3.0 mm ID HALO column to a larger ID HALO column for use on an HPLC system, the same recom-mendations apply for adjusting flow rate and injection volume as just described above for isocratic methods. Once again, to keep the relative retention comparable and to minimize band broadening for early-eluting analytes, an injection delay is used to adjust the effective delay volume of the HPLC system to match that of the UHPLC system. Example 2 will again illustrate this.

example 2

In this second example, a gradient UHPLC method using a 2.1 × 50 mm HALO column will be transferred to an HPLC system using 50-mm long 3.0-and 4.6-mm ID HALO columns. These two situations correspond to Scenarios B and C of Figure 1. The conditions for the UHPLC gradient method are shown in Table 4.

The flow rate for the 2.1 × 50 mm column is 0.42 mL/min at 40°C, with acetonitrile and water with 0.1% formic acid as additive in both solvents, with a 1-μL injection. Gradient is from 3% ACN to 70% in 2.7 minutes with a short hold until 3.5 min. The gradient returns to 3% ACN at 4.0 min., and equilibrates at 3% for 1.6 minutes (5 V

m + 2 V

D).

One can calculate the %B at the column by noting that the delay volume of 0.120 mL will impose a 0.29 min delay time (0.120 mL/0.42 mL/min), so that the %B at the column will not reach 70% until 2.70 + 0.29 = 2.99 min. By inspection, you can deter-mine the 3.79-min and 5.89-min time points for the %B at the column. The final stop time for the gradient program is then set at a time longer than 5.89 min. (6.0 min), so that the run ends when the desired equilibration time at the column occurs. Usually, there is enough time in the autosampler cycle time to provide additional equilibration time, but the projected gradient program for the HPLC system (Table 4) shows how important it can be to consider and build in this additional equilibration time at the column.

To implement this method on a 3.0 × 50 mm HALO column on an HPLC system (0.8 mL delay volume), the injection volume and flow rate must be scaled to keep the loading and linear velocity the same as on the 2.1 × 50 mm column.

Vinj = 1.0 μL × (3.0/2.1)2 = 2.0 μL

Flow rate, F = 0.42 mL/min × (3.0/2.1)2 = 0.86 mL/min

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Table 4: Comparison of Gradient Programs and %B at Column for UHPLC and HPLC Methods using 2.1 and 3.0 mm ID HALO Columns

For the HPLC system, we calculate the %B at column for the various gradient time points as above. It thus takes 0.93 min. (0.8 mL/0.86 mL/min) for the beginning of the gradient program to reach the column. It takes 2.70 + 0.93 min. = 3.63 min. for the 70% B composition mobile phase to reach the column, and the 70% B composition hold at the column lasts until 3.63 + (3.50 – 2.70) = 4.43 min. The original return to 3% B takes 0.5 min. which takes until 4.43 + 0.5 = 4.93 min. at the column.

For the HPLC method using the 3.0 × 50 mm column, a much longer hold time in the gradient program is needed to re-equilibrate the column (hold at 3%B from 4.0 to 6.9 min.) because of the desired equilibration time1 ([5 Vm + 2 V

D]/0.86 mL/

min = [5 × 0.177 + 2 × 0.8]/0.86 = 2.9 min.) This 2.9-min hold is added on to the 4.93-min time point (%B at column), which requires a method stop time of ~8.0 min. (instead of 6.9 or 7.0 min.), if one does not consider the lag in %B at the column.1Note: Schellinger et al. have reported that “excellent repeatability (±0.002 min in retention time) is achieved with at most 2 column volumes of re-equilibration, whereas full equilibration can require considerably more than 20 column volumes.” (See Reference 4.)

Time %B Time %B (column)

0.00 3 0 3

2.70 70 0.29 3

3.50 70 2.99 70

4.00 3 3.79 70

5.60 3 4.29 3

6.00 Stop 5.89 3

Time %B Time %B (column)

0.00 3 0.00 3

2.70 70 0.93 3

3.50 70 3.63 70

4.00 3 4.43 70

6.90 3 4.93 3

8.00 Stop 7.83 3

Ving 1.0 µL

VD 0.120 mL

ID 2.1 mm

Length 50 mm

VM 0.087 mL

F 0.42 mL/min

Linear Velocity (µ) 4.04 mm/sec

Ving 2.0 µL

VD 0.800 mL

ID 3 mm

Length 50 mm

VM 0.177 mL

F 0.86 mL/min

Linear Velocity (µ) 4.04 mm/sec

uHplC SySTem HplC SySTem

MAC-MOD ANALYTICAL TECHNICAL REPORT | 8

Delay time 0.93 min.

Injection Delay 0.79 min.

Corrected Delay time 0.14 min.

Effective Delay volume 0.12 mL

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Effective Delay Volume Adjustment Using an Injection Delay

Finally, to adjust the effective delay volume on the HPLC to match the delay volume of the UHPLC system, an appropriate injection delay should be implemented on the HPLC (see Table 4).

Injection Delay (HPLC) = (VD, HPLC – VD, UHPLC)/FHPLC = (0.80 mL – 0.12 mL)/0.86 mL/min = 0.79 min.

Similar calculations for Scenario C (Figure 1) for the transfer of the UHPLC method using the 2.1 × 50 mm HALO column to a 4.6 × 50 mm HALO column on the HPLC system are shown in Table 5. For the much faster scaled flow rate of 2.0 mL/

min on the 4.6-mm ID column, the injection delay needs to be only 0.34 min., and the scaled injection volume is 4.8 μL, or a nominal 5 μL.

The resulting chromatograms from the transfer of a gradient method for 11 phenols from a 2.1 × 50 mm HALO column on a UHPLC system to a 4.6 × 50 mm HALO column on an HPLC system are shown in Figure 5. Corresponding chromatograms for the transfer of the UHPLC method developed on a 2.1 mm ID column to 2.1 and 3.0 mm HALO columns on an HPLC system are presented in Figure 6.

Time %B Time %B (column)

0.00 3 0 3

2.70 70 0.29 3

3.50 70 2.99 70

4.00 3 3.79 70

5.60 3 5.89 3

6.00 Stop

Time %B Time %B (column)

0.00 3 0.00 3

2.70 70 0.40 3

3.50 70 3.10 70

4.00 3 3.90 70

5.82 3 4.40 3

6.50 Stop 6.22 3

Ving 1.0 µL

VD 0.120 mL

ID 2.1 mm

Length 50 mm

VM 0.087 mL

F 0.42 mL/min

Linear Velocity (µ) 4.04 mm/sec

Delay time 0.286 min.

Delay volume 0.12 mL

Delay time 0.40 min.

Injection Delay 0.34 min.

Corrected Delay time 0.060 min.

Effective Delay volume 0.12 mL

Ving 4.8 µL

VD 0.800 mL

ID 4.6 mm

Length 50 mm

VM 0.415 mL

F 2.02 mL/min

Linear Velocity (µ) 4.04 mm/sec

uHplC SySTem HplC SySTem

Table 5: Comparison of Gradient Programs and %B at Column for UHPLC and HPLC Methods using 2.1 and 4.6 mm ID HALO Columns

ReferencesBridging the Gap between UHPLC and HPLC: Easy Method Transfer Using Fused-Core® Columns, S. A. Schuster and T. J. Waeghe, PittCon 2012 poster.

Technical Report: How to Measure and Reduce HPLC Equipment Extracolumn Volume, MAC-MOD Analytical, Inc., www.mac-mod.com .

Technical Report: Quick Tips for Converting Conventional Reversed-Phase HPLC Separations to Ultra-Fast Separations, MAC-MOD Analytical, Inc., www.mac-mod.com.

High speed gradient elution reversed-phase liquid chromatography, Adam P. Schellinger, Dwight R. Stoll, Peter W. Carr; Journal of Chromatography A, 1109 (2006) 253–266.

A practical approach to transferring linear gradient elution methods, Adam P. Schellinger, Peter W. Carr, Journal of Chromatography A, 1077 (2005) 110–119.

Method transfer for fast liquid chromatography in pharmaceutical analysis. Application to short columns packed with small particle. Part I: isocratic separation, D. Guillarme, D.T.T. Nguyen, S. Rudaz, J.L. Veuthey, Eur. J. Pharm. Sci. 66 (2007) 475–482

Method transfer for fast liquid chromatography in pharmaceutical analysis: Application to short columns packed with small particle. Part II: Gradient experiments, D. Guillarme, D.T.T. Nguyen, S. Rudaz, J.L. Veuthey, European Journal of Pharmaceutics and Biopharmaceutics, 68 (2008) 430–440.

Maximizing peak capacity and separation speed in liquid chromatography, Patrik Petersson, Andre Frank, James Heaton, Melvin R. Euerby, J. Sep. Sci., 2008, 31, 2346–2357.

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Gradient was from 3 to 70% acetonitrile/water with 0.1% formic acid in both solvents in 2.7 minutes. Injection delay for the quaternary HPLC was set at 0.41 min. The flow rates and injection volume for the 4.6 mm ID column were scaled as described. UHPLC and HPLC delay volumes were measured to be 0.12 and 0.70 mL, respec-tively. Data rate was 40 Hz for the UHPLC system and 13.7 Hz for the HPLC system. Response times were 0.1 and < 0.125 sec., respectively, for the UHPLC and HPLC systems. Flow cells were 2 μL and 5 μL for the UHPLC and HPLC systems, respectively.

Sample analytes, in elution order, are: hydroquinone, resorcinol, catechol, phenol, 4-nitrophenol, 4,4’-biphenol, 2-chlorophenol, 4-chlorophenol, 2,2’-biphenol, 2,6–dichlorophenol, and 2,4-dichlorophenol

Comparable separations are produced when transferring a method developed on a UHPLC system on the 2.1 × 50 mm HALO column to an HPLC system using the same 2.1 mm ID column and using a 3.0 × 50 mm HALO column (Figure 6). Flow rates and injection volumes were identical for the HPLC system with the 2.1 mm ID column, but were scaled as described for the 3.0 mm ID column. Injection delays were set at 1.74 and 0.93 min, respectively, for the 2.1 and 3.0 mm ID HALO columns on the HPLC system. Other analysis conditions were the same as those described for Figure 5.

FIgure 5: Comparison of Chromatograms Generated on a UHPLC System and an HPLC System When Transferring a Method Using Different HALO Column IDs

FIgure 6: Chromatograms from a UHPLC Method Transferred to an HPLC System Using the Original 2.1 mm ID HALO Column and a 3.0 mm ID HALO Column

uHplC SySTem

HplC SySTem

0.0 1.0 2.0 2.0 3.0 4.0 5.0

2.0 3.00.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6

200

100

0

400

200

0

600

400

200

0

600

400

200

0

Time (min) Time (min)

Time (min)Time (min)

mAU mAU

mAUmAU

Note: An injection delay keeps relative retention of analytes comparable, but absolute retention times will vary.

Agilent 1200SL0.42 mL/min, 1 µL inj. No Injection Delay

Agilent 1100 Quaternary0.42 mL/min, 1 µL inj. Injection Delay 1.74 min.

Agilent 1100 Quaternary0.86 mL/min, 2 µL inj. Injection Delay 0.93 min.

Agilent 1100 Quaternary2.0 mL/min, 4.8 µL inj. Injection Delay 0.41 min.

2.1 x 50 mm HalO C182.1 x 50 mm HalO C18

3.0 x 50 mm HalO C184.6 x 50 mm HalO C18

MAC-MOD ANALYTICAL TECHNICAL REPORT | 10

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