improve the chromatographic performance of your assay...

Improve the chromatographic performance of your assay without changing Improve the chromatographic performance of your assay without changing

method or HPLC equipment

L. Pereira, T. Edge, H. Ritchie, S. Luke

Thermo Fisher Scientific, Manor Park, Runcorn, UK, WA7 1TThermo Fisher Scientific, Manor Park, Runcorn, UK, WA7 1T

Overview ResultsOverview

Purpose: to compare the chromatographic performance of

solid core 4 µm particle packed columns with that of fully

porous 5 and 3 µm particle packed columns.

Efficiency and Pressure

FIGURE 1: Efficiency comparison using Van Deemter plots

for Accucore XL 4 µm and fully porous 5 and 3 µm.

Methods: Isocratic and gradient methods that allow

comparison of chromatographic parameters such as

efficiency, resolution, sensitivity, column backpressure and

for Accucore XL 4 µm and fully porous 5 and 3 µm.

14.0

16.0

18.0

analysis time.

Results: The solid core 4 µm particles provide significant

improvements over fully porous 5 and 3 µm in terms of

separation efficiency, resolution, and sensitivity, with only a

8.0

10.0

12.0

14.0

Pla

te h

eig

ht

(H,µ

m)

separation efficiency, resolution, and sensitivity, with only a

small increase or a decrease in column backpressure for the

5 and 3 µm respectively.

0.0

2.0

4.0

6.0

Pla

te h

eig

ht

(H,

Introduction

In the past decade there has been continuous drive to

0.0

0.0 0.5 1.0 1.5

Accucore XL 4µm

In the past decade there has been continuous drive to

develop chromatographic stationary phases to perform high

efficiency HPLC. One such area is the use of solid core

materials which has gained a lot of interest from

FIGURE 2: Comparison of column pressure for Accucore XL

4 µm and fully porous 5 and 3 µm (150x4.6 mm columns).materials which has gained a lot of interest from

manufacturers and also end users. This type of packing

material has a solid core surrounded by a porous outer layer,

which allows for more uniform particle sizing. When these

4 µm and fully porous 5 and 3 µm (150x4.6 mm columns).

200

250

which allows for more uniform particle sizing. When these

partially porous particles were introduced, the diameter would

typically be between 2 and 3 µm, and these particles provide

similar performance to smaller sub-2 µm fully porous 100

150

Pre

ssu

re (

ba

r)

particles at significantly lower column backpressures.

The very tight particle size distribution of solid core materials

results in columns with high permeability, and therefore for 0

50

Pre

ssu

re (

ba

r)

results in columns with high permeability, and therefore for

the same nominal pressure they give better separations than

fully porous materials. Often however, solid core particle

packed columns are narrow-bore and short which has

0

200 400 600 800

Accucore XL 4µmpacked columns are narrow-bore and short which has

implications in terms of the system set-up. System dead

volume and operating parameters have to be optimised in

order to get the best possible performance out of the column.

On average (across the flow rate range tested) Accucore XL

4 µm gives:order to get the best possible performance out of the column.

The work presented in this poster demonstrates that by using

a new solid core 4 µm particle, packed in conventional

column dimensions, significant improvements in the assay

75% more efficiency than fully porous 5

efficiency than fully porous 3

42% higher pressure than fully porous 5 column dimensions, significant improvements in the assay

performance can be achieved without the need to make

changes to the operating parameters or system set-up. With

the Thermo Scientific Accucore XL 4 µm columns it is FIGURE 3: Performance comparison of Accucore XL 4 µm

lower pressure than fully porous 3

the Thermo Scientific Accucore XL 4 µm columns it is

possible to dramatically improve separation efficiency,

resolution, sensitivity over those obtained with conventional

fully porous 5 and 3 µm particle packed columns. These

1.E+04

t0 (s)

FIGURE 3: Performance comparison of Accucore XL 4 µm

and fully porous 5 and 3 µm using kinetic plots: efficiency

per unit time.

fully porous 5 and 3 µm particle packed columns. These

improvements are obtained with only 40% increase in

backpressure over the 5 µm and a reduction over the 3 µm.

Some reduction in analysis time is also possible.

1.E+03

Some reduction in analysis time is also possible.

1.E+02

Methods

Liquid Chromatography

1.E+01

Columns: Accucore XL C18 4 µm, 150x4.6 mm

Fully porous C18 5 µm, 150x4.6 mm

Fully porous C18 3 µm, 150x4.6 mm

1.E+00

1.E+04

Accucore XL 4µm

FIGURE 4: Performance comparison of Accucore XL 4 µm Fully porous C18 3 µm, 150x4.6 mm

1. Efficiency and pressure testing:

Mobile phase: water / ACN (50:50)

FIGURE 4: Performance comparison of Accucore XL 4 µm

and fully porous 5 and 3 µm using kinetic plots: column

impedance (E) relative to linear velocity (u).1.E+05Mobile phase: water / ACN (50:50)

Flow rates: 0.4 to 2.0 mL/min (every 0.1 mL/min)

Temperature: 30 °C

Imp

ed

an

ce

Detection: UV at 254nm (0.1s rise time, 20 Hz)

Injection volume: 1 µL

Test probe: o-Xylene (t0 marker – theophylline).

1.E+04

Test probe: o-Xylene (t0 marker – theophylline).

2. Resolution, sensitivity and productivity testing:

Mobile phase: A – water; B – ACN

Temperature: 30 °C

Detection: UV at 247 nm (0.1s rise time, 20 Hz)

1.E+03

0

Accucore XL 4µm

Injection volume: 5 µL

Sample: 1. Uracil, 2. Tebuthiuron; 3. Metoxuron; 4. Monuron;

5. Chlorotoluron; 6. Diuron; 7. Linuron

Impedance is a term that gives a true measure of the

performance of the column as it incorporates efficiency, time

and pressure, which are critical parameters of a separation.

Accucore XL 4µm

• Standard gradient: 35 to 60% B in 10 min

Flow rate: 1.0 mL/min

• Scaled gradient: 35 to 60% B in 7.5 min


Lower impedance values indicate faster and higher efficiency

separations performed at lower backpressures.

On average (across the flow rate range tested) Accucore XL • Scaled gradient: 35 to 60% B in 7.5 min


(optimum flow from van Deemter plot)


4 µm gives:

59% more efficiency per unit time than fully porous 5

and 53% more efficiency per unit time than fully porous 3 (optimum flow from van Deemter plot)

• Optimized gradient: 35 to 60% B in 4 min


and 53% more efficiency per unit time than fully porous 3

(Figure 3).

79% lower impedance than fully porous 5

lower impedance than fully porous 3 lower impedance than fully porous 3

Improve the chromatographic performance of your assay without changing Improve the chromatographic performance of your assay without changing

Thermo Fisher Scientific, Manor Park, Runcorn, UK, WA7 1TThermo Fisher Scientific, Manor Park, Runcorn, UK, WA7 1T

Efficiency comparison using Van Deemter plots

for Accucore XL 4 µm and fully porous 5 and 3 µm. FIGURE 5: Comparison of the resolution of a critical pair

(peaks 2 and 3) on fully porous 5 and 3 µm and Accucore

Resolution, Sensitivity and Productivity

for Accucore XL 4 µm and fully porous 5 and 3 µm. (peaks 2 and 3) on fully porous 5 and 3 µm and Accucore

XL 4 µm. Standard gradient conditions.

Fully porous 5 µmRs = 2.47

Fully porous 3 µmRs = 2.82

Accucore XL 4 µmRs = 3.13

1.5 2.0 2.5 3.0 3.5 4.0 4.5

Linear velocity u (mm/s)

Fully porous 5µm Fully porous 3µm Minutes0 1 2 3 4 5 6 7 8 9 10

Comparison of column pressure for Accucore XL

4 µm and fully porous 5 and 3 µm (150x4.6 mm columns).

FIGURE 6: Comparison of average signal-to-noise ratios

(S/N) on fully porous 5 and 3 µm and Accucore XL 4 µm

for 1 ng of each solute loaded on column. Scaled 4 µm and fully porous 5 and 3 µm (150x4.6 mm columns). for 1 ng of each solute loaded on column. Scaled

gradient conditions.

Fully porous 5 µmS/N = 4.9

Fully porous 3 µm

S/N = 10.6

S/N = 5.3

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Accucore XL 4 µmS/N = 10.6

1000 1200 1400 1600 1800 2000

Flow rate (µL/min)

Fully porous 5µm Fully porous 3µm

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

FIGURE 7: Productivity comparison for fully porous 5 µm

and Accucore XL 4 µm.


Fully porous 5 µm

Rs = 2.47 Rt = 8.62

Standard gradientRs = 3.13

75% more efficiency than fully porous 5 µm and 50% more

efficiency than fully porous 3 µm (Figure 1).

42% higher pressure than fully porous 5 µm and 13% Rs = 3.02

Standard gradientRs = 3.13

Rt = 6.56

Accucore XL 4 µm

Standard gradient

Performance comparison of Accucore XL 4 µm

lower pressure than fully porous 3 µm (Figure 2).

Accucore XL 4 µm

Rs = 2.99

Rs = 3.02

Rt = 5.07

Scaled gradient


and fully porous 5 and 3 µm using kinetic plots: efficiency

Accucore XL 4 µm

Rs = 2.99Rt = 4.06

Optimized gradient

The higher efficiencies of the solid core 4 µm material over

the fully porous 5 and 3 µm materials results in improved

Minutes0 1 2 3 4 5 6 7 8 9 10

the fully porous 5 and 3 µm materials results in improved

resolution and sensitivity:

27 and 11% higher resolution of the critical pair (Figure 5)

119 and 100% higher signal/noise ratios (Figure 6)119 and 100% higher signal/noise ratios (Figure 6)

The higher efficiencies of the Accucore XL 4 µm over a wide

flow rate range can be used to reduce analysis time by

optimizing flow rate and gradient. In Figure 7, a reduction of Efficiency (/) optimizing flow rate and gradient. In Figure 7, a reduction of

run time by half is demonstrated, still with improved

resolution of the critical pair.

1.E+05


Efficiency (/)


Conclusion

The new Accucore XL 4 µm solid core columns provide


and fully porous 5 and 3 µm using kinetic plots: column

impedance (E) relative to linear velocity (u).The new Accucore XL 4 µm solid core columns provide

improvements in efficiency in excess of 50% over fully porous

5 and 3 µm columns

The backpressure of the new Accucore XL 4 µm solid core η20

N

PtE

∆=

columns is moderately higher than fully porous 5 µm, but

lower than fully porous 3 µm

The new Accucore XL 4 µm solid core columns exhibit

η2N

significantly lower impedance than fully porous materials

Significant improvements in resolution and signal-to-noise

ratios were demonstrated with the Accucore XL 4 µm material

The Accucore XL 4 µm columns produce the improvements

listed above with no changes to methodology or HPLC

system set-up. The transfer to Accucore XL 4 µm is quick and

easy

3


u0 (mm/s)

easy

Impedance is a term that gives a true measure of the

performance of the column as it incorporates efficiency, time

and pressure, which are critical parameters of a separation. Additional information



Lower impedance values indicate faster and higher efficiency

separations performed at lower backpressures.


Additional information

For additional information, please visit our Chromatography Resource Centre which

can be found at: www.thermoscientific.com/CRC

©2012 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property On average (across the flow rate range tested) Accucore XL

59% more efficiency per unit time than fully porous 5 µm

and 53% more efficiency per unit time than fully porous 3 µm

of Thermo Fisher Scientific Inc. and its subsidiaries . Specifications, terms and pricing

are subject to change. Not all products are available in all countries. Please consult

your local sales representatives for details.

PSCCS 1512 0812and 53% more efficiency per unit time than fully porous 3 µm

79% lower impedance than fully porous 5 µm and 72%

lower impedance than fully porous 3 µm (Figure 4).lower impedance than fully porous 3 µm (Figure 4).

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