Maximizing the Peak-Capacity-to-Analysis-Time Ratio in One-Dimensional Liquid Chromatography

Michael Heidorn, Susanne Fabel and Frank Steiner Thermo Fisher Scientific, Germering, Germany

2 Maximizing the Peak-Capacity-to-Analysis-Time Ratio in One-Dimensional Liquid Chromatography

Maximizing the Peak Capacity to Analysis Time Ratio in One DimensionalMaximizing the Peak-Capacity-to-Analysis-Time Ratio in One-Dimensional a g t e ea Capac ty to a ys s e at o O e e s o aCLiquid ChromatographyLiquid Chromatography

Mi h l H id S F b l d F k St iMichael Heidorn, Susanne Fabel and Frank SteinerMichael Heidorn, Susanne Fabel and Frank SteinerTh Fi h S i tifi G i GThermo Fisher Scientific, Germering, GermanyThermo Fisher Scientific, Germering, Germany

Overview Results While the theoretical plate heights (Figure 3) of both column-Overview Results p g ( g )series merged each other the same way as it was already

Purpose: A separation of complex sample mixtures with Comparison of 2 1 × 100 mm Columnsseries merged each other the same way as it was already seen in figure 1 the number of theoretical plates (Figure 4)Purpose: A separation of complex sample mixtures with

appropriate resolution in the shortest time possibleComparison of 2.1 × 100 mm Columns seen in figure 1, the number of theoretical plates (Figure 4)

was much higher for the longer sequence of Accucoreappropriate resolution in the shortest time possible. Different chromatographic columns (porous C18-silica, was much higher for the longer sequence of Accucore columns The maximum peak capacity of the AccucoreMethods: Different stationary phase architectures, column

g (fused-core silica-based C18, and monolithic silica-based columns. The maximum peak capacity of the Accucore

column chain was found to be n 220 at a retention time ofy p ,lengths, and temperatures, as well as applying pressures

,C18e) were tested by using the RP-5 standard under column chain was found to be nc = 220 at a retention time of

b t t 40 i (Fi 5) Th i k it flengths, and temperatures, as well as applying pressures up to 1200 bar

C18e) were tested by using the RP 5 standard under isocratic conditions (Figure 1 and Figure 2) about tR = 40 min (Figure 5). The maximum peak capacity of up to 1200 bar. isocratic conditions (Figure 1 and Figure 2). the Hypersil GOLD column was probably not achieved but

Results: Exploiting the maximum column pressure, the FIGURE 1: Efficiencies of columns with different found to be approximately nc = 150 at a retention time of p g p ,best peak-capacity-to-analysis-time ratio for isocratic stationary phase architectures but the same

y cabout tR = 25 min (Figure 5). The flow-pressure range was p p y y

conditions was determined by Thermo Scientific™y p

dimension of 2.1 × 100 mm.R ( g ) p g

similar for both column-series (Figure 6).conditions was determined by Thermo ScientificAccucore™ columns 2 6 μm particle size With gradient

dimension of 2.1 100 mm.Poro s Particles d 2 5 m C18 Comp Wm

)

similar for both column series (Figure 6).Accucore columns, 2.6 μm particle size. With gradient conditions Thermo Scientific™ Hypersil GOLD™ columns

Porous Particles, dp= 2.5 μm, C18, Comp. WPorous Particles d = 1 9 μm C18 Hypersil GOLD19(µ

m Traditional Chinese Medicine (TCM)conditions, Thermo Scientific Hypersil GOLD columns, 1 9 μm particle size were found to be superior

Porous Particles, dp= 1.9 μm, C18, Hypersil GOLDSolid Core Particles, dp= 2.6 μm, C18, Accucore17H

(

The series of Hypersil GOLD (2 1 × 300 mm) columns and1.9 μm particle size, were found to be superior. Solid Core Particles, dp 2.6 μm, C18, AccucoreSolid Core Particles, dp= 2.7 μm, C18, Comp. A15

17

ght The series of Hypersil GOLD (2.1 300 mm) columns and

Accucore (2 1 × 450 mm) columns were tested by using the

I t d tip μ p

Monolithic Rod, C18(e), Comp. M1315

eig

ne

Accucore (2.1 × 450 mm) columns were tested by using the TCM sample “Dang Gui Long Hui Wan“ under gradientIntroduction 13

e H

eyl

en TCM-sample Dang Gui Long Hui Wan under gradient conditions Applying the optimum linear velocity to each the

Th ti f ll l d ith i t11late

o-Xy conditions. Applying the optimum linear velocity to each, the

th A l i i t d k itThe separation of all sample compounds with appropriate 9lPl

ofo three Accucore columns in series generated a peak capacity

resolution in the shortest time possible is a common goal 7ical o of nc = 670 at the gradient time tg = 60 min with a

of most chromatographers. This goal becomes even more 5reti g

backpressure of 850 bar. The two Hypersil GOLD columns in g grelevant with highly complex samples. Generally, highest 3

5

eor

series generated a peak capacity of nc = 480 at the gradient g y p p y, gpeak capacities are achieved by employing an additional

30.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5Th

e g p p y c gtime tg = 28 min with a backpressure of 1200 bar.peak capacities are achieved by employing an additional

chromatographic dimension Dedicated instrumentation Linear Velocity u (mm/s)time tg 28 min with a backpressure of 1200 bar.

chromatographic dimension. Dedicated instrumentation, extensive method development and a proper combination FIGURE 2: Backpressures of columns with different FIGURE 7: Calculated peak capacities with columnextensive method development, and a proper combination of separation chemistries are necessary in this case The

FIGURE 2: Backpressures of columns with different t ti h hit t b t th

FIGURE 7: Calculated peak capacities with column chains in gradient mode: Hypersil GOLD 2 1 × 300 mmof separation chemistries are necessary in this case. The

theoretical basis has been summarized by Guiochon 1stationary phase architectures but the same chains in gradient mode: Hypersil GOLD 2.1 × 300 mm

l A 2 1 × 450 ltheoretical basis has been summarized by Guiochon.1 dimension of 2.1 × 100 mm. column or Accucore 2.1 × 450 mm column.

Highest resolution in shortest time can also be achieved Porous Particles d = 2 5 μm1000 700Highest resolution in shortest time can also be achieved by maximizing the peak-capacity-to-analysis-time ratio of

Porous Particles, dp 2.5 μmPorous Particles, dp= 1.9 μm F 0 45 0 65 0 60 0 45600

700

yby maximizing the peak-capacity-to-analysis-time ratio of an application This approach is easily implemented by

, p μSolid Core Particles, dp= 2.7 μm800) (mL/min)

0.45 0.65 0.60 0.45500600

city

an application. This approach is easily implemented by applying columns of extended length packed with porous

pSolid Core Particles, dp= 2.6 μmM lithi R dba

r)

tG 40 42 28 60500pa

c

applying columns of extended length packed with porous b 2 i ti l O th th h d h t

Monolithic Rod600e(b tG

(min)40 42 28 60400C

a

sub-2-micron particles. On the other hand, such a setup ure ( )

Pak

lead to a column head pressure up to 1200 bar and even 400ess P

(bar)930 1200 1200 850200P

e

more. In this work, experimental results from the use of Max Pressure of MonolithPr

e (bar)

n 430 600 480 670different stationary phase architectures in column lengths Max. Pressure of Monolith200

P nc 430 600 480 6700y p g

of 100 mm to 450 mm and at pressures up to 1200 bar are0

of 100 mm to 450 mm and at pressures up to 1200 bar are discussed Furthermore it is evaluated which approach 0 FIGURE 8: Ratio of peak-capacity-to-analysis-time with discussed. Furthermore, it is evaluated which approach provides the best peak-capacity-to-analysis-time ratio for

0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05Fl t F ( L/ i )

p p y ycolumn chains in gradient mode: Hypersil GOLD 2.1 ×provides the best peak-capacity-to-analysis-time ratio for

the analysis of complex sample mixturesFlow rate F (mL/min) g yp

300 mm column or Accucore 2.1 × 450 mm column.the analysis of complex sample mixtures. In one dimensional liquid chromatography the best peak

300 mm column or Accucore 2.1 450 mm column.17

M th dIn one-dimensional liquid chromatography, the best peak-

it t l i ti ti i hi d b l iti17

o FMethods capacity-to-analysis-time ratio is achieved by exploiting 14to

Rat

io F (mL/min)

0.45 0.65 0.60 0.45

Liquid Chromatography System the flow-pressure footprint of the system and column 11ci

ty

e R (mL/min)

/tLiquid Chromatography System™ ™ ™

hardware. High peak capacity is basically achieved 11

pac

Tim

e nc/tG( / )

11 14 17 11Thermo Scientific ™ Dionex™ UltiMate™ 3000 XRS system: through a high number of theoretical plates (N) per C

ap s T (1/min)

solvent rack SR-3000, pump LPG-3400XRS, sampler g g p ( ) p

column. The optimum linear velocity is determined by the 5ak C

lysi P 930 1200 1200 850p p pWPS-3000TXRS, column thermostat TCC-3000RS,

column. The optimum linear velocity is determined by the minimum theoretical plate height (Figure 1) Extending the P

eaA

nal

(bar)930 1200 1200 850

, ,and detector DAD-3000RS.

minimum theoretical plate height (Figure 1). Extending the column length means enhancing the column efficiency (N) 0

Aand detector DAD 3000RS. column length means enhancing the column efficiency (N) but also requires a higher pressure to achieve the same

0

Data Analysis but also requires a higher pressure to achieve the same linear velocity (Figure 2) The Accucore column chain provided higher peak capacities

Thermo Scientific™ Dionex™ Chromeleon™linear velocity (Figure 2). The Accucore column chain provided higher peak capacities

th th H il GOLD l h i t h fl tThermo Scientific Dionex ChromeleonChromatograph Data S stem Soft are ersion 6 8 Different concepts of stationary phase design were than the Hypersil GOLD column chain at each flow rate Chromatography Data System Software version 6.8 p y p g

compared by using identical column dimensions. The (Figure 7). The ratio between the peak capacities of both Comparison of 2 1x100 mm columns

p y gmonolithic rod showed the poorest efficiency but also column chains was the same as found under isocratic Comparison of 2.1x100 mm columns

C l Diff t d i f t ti h ( llmonolithic rod showed the poorest efficiency but also required the lowest flow-pressure footprint The maximum conditions by using phenones (Figure 5). But when

Columns: Different designs of stationary phase (all required the lowest flow-pressure footprint. The maximum operation pressure of a monolithic rod is about 200 bar

y g p ( g )comparing the ratio of peak capacity to analysis time, the

RP-C18) with 2.1 × 100 mm each operation pressure of a monolithic rod is about 200 bar and was almost reached for the 2 1 x 100 mm column at a

comparing the ratio of peak capacity to analysis time, the Accucore column chain generated 14 peaks per minute while

RP-5 Standard: Theophylline, p-nitroaniline, phenetole, and was almost reached for the 2.1 x 100 mm column at a fl t f 1 L/ i (Fi 2) H li

Accucore column chain generated 14 peaks per minute while the Hypersil GOLD column chain generated 17 peak per

o-xylene, and methyl benzoate flow rate of 1 mL/min (Figure 2). Hence, coupling li hi d h i hi h ld h l

the Hypersil GOLD column chain generated 17 peak per minute at 1200 bar backpressure (Figure 8) Thus byy , y

Mobile Phase A: Fisher Scientific™ Optima™ LC/MS water monolithic rods to a chain which would show an equal minute at 1200 bar backpressure (Figure 8). Thus, by exhausting the maximum pressure the Accucore chain wasMobile Phase A: Fisher Scientific Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrile number of theoretical plates compared to the other column exhausting the maximum pressure, the Accucore chain was bl t hi th hi h t k it b t th H ilMobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% B types is not feasible. able to achieve the highest peak capacity but the Hypersil OIsocratic Conditions: 50% A and 50% B

Flow Rate: 0 1 1 0 mL/minypHypersil GOLD and Accucore columns were found to

GOLD column chain provided the maximum ratio of peak Flow Rate: 0.1–1.0 mL/minInjection Volume:1 µL

Hypersil GOLD and Accucore columns were found to provide the highest column efficiency (Figure 1)

capacity to analysis time.Injection Volume:1 µLC l T t 30 °C

provide the highest column efficiency (Figure 1). F th th A l h d b t 60% fColumn Temperature: 30 °C Furthermore, the Accucore columns showed about 60% of th b k l ti t th H il GOLD l ConclusionDetection wavelength: 254 nm the backpressure relative to the Hypersil GOLD columns Conclusion

Detection Rate: 20 Hz at identical flow rates (Figure 2). Accordingly, to exploit the By exploiting the maximum pressure of the column Comparison of columns coupled in series flow-pressure footprint of the system and column

y p g phardware, a 2.1 × 450 mm long chain of Accucore C18Comparison of columns coupled in series

C l S i H il GOLD C18

p p yhardware, three Accucore 2.1 × 150 mm columns were

hardware, a 2.1 450 mm long chain of Accucore C18 (2 6 μm) columns could be built It produced a higherColumn-Series: Hypersil GOLD C18, ,

coupled in series to a sequence of 2.1 × 450 mm. For(2.6 μm) columns could be built. It produced a higher efficiency (N) than the 2 1 × 300 mm long Hypersil GOLD1.9 μm, 2.1 × 200 and 2.1 × 100 mm coupled in series to a sequence of 2.1 450 mm. For

Hypersil GOLD columns 2 1 × 200 mm and 2 1 × 100efficiency (N) than the 2.1 × 300 mm long Hypersil GOLD (1 9 μm) column chainAccucore C18, Hypersil GOLD columns , 2.1 × 200 mm and 2.1 × 100

mm were coupled to a sequence of 2 1 × 300 mm(1.9 μm) column chain.

2.6 μm, 2.1 × 150 mm (three) mm were coupled to a sequence of 2.1 × 300 mm. On the Accucore 2.1 × 450 mm column chain, a peakμ , ( )

Sample: Phenones and uracil Comparison of Columns Coupled in SeriesOn the Accucore 2.1 450 mm column chain, a peak capacity of 600 was achieved for the TCM “Dang GuiSample: Phenones and uracil

Mobile Phase A: Optima LC/MS waterp p

The series of Hypersil GOLD (2 1 × 300 mm) andcapacity of 600 was achieved for the TCM Dang Gui Long Hui Wan” with 42 min gradient time (14 peaks perMobile Phase A: Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrileThe series of Hypersil GOLD (2.1 × 300 mm) and Acc core (2 1 × 450 mm) col mns ere characteri ed

Long Hui Wan with 42 min gradient time (14 peaks per minute) A peak capacity of 670 could be realized with 60Mobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% BAccucore (2.1 × 450 mm) columns were characterized

ith lk l h t d d d i ti ditiminute). A peak capacity of 670 could be realized with 60

i di t ti hi h i d th k itIsocratic Conditions: 50% A and 50% BFlow Rate: 0 05 0 5 mL/min

with alkylphenone standard under isocratic conditions. min gradient time, which compromised the peak-capacity-t l i ti ti (11 k i t )Flow Rate: 0.05–0.5 mL/min

I j ti V l 5 L FIGURE 4: TheoreticalFIGURE 3 Theoretical to-analysis-time ratio (11 peaks per minute).Injection Volume: 5 μL FIGURE 4: Theoretical

plate numbers (N) ofFIGURE 3. Theoretical plate heights (H) of On the Hypersil GOLD 2 1 × 300 mm column chain aColumn Temperature: 35 °C plate numbers (N) of

hexanophenoneplate heights (H) of hexanophenone

On the Hypersil GOLD 2.1 × 300 mm column chain, a peak capacity of 430 was achieved for the TCM “DangDetection: 250 nm hexanophenone.hexanophenone. peak capacity of 430 was achieved for the TCM Dang G i L H i W “ i 40 i di t ti (11 kDetection Rate: 20 Hz N 100 x10³20m

)

Hypersil GOLD 2 1 x 300 mmGui Long Hui Wan“ in 40 min gradient time (11 peaks per

i t ) A k it f 480 d t i d ith 28T diti l Chi M di i (TCM) es 80

20

(µm

A 2 1 450

Hypersil GOLD 2.1 x 300 mm minute). A peak capacity of 480 was determined with 28 Traditional Chinese Medicine (TCM)

plat 80

16H ( Accucore 2.1 x 450 mm min gradient time, which benefited the peak-capacity-to-

Columns: see above

ofp

6016

ght analysis-time ratio (17 peaks per minute).

TCM-Sample: Dang Gui Long Hui Wan Nr. 3560 B, ero

4010Hei

g y ( p p )

M lithi l t id hi h k itiHerba Natura Harald Noll BV, Dinxperlo, the Netherlands mbe 4010

e H Monolithic columns cannot provide high peak capacities

i bl d h t ti d t th i i, p ,

Sample Preparation: 200 mg of sample mixed with 10 Num 204la

te in a comparable and short time due to their maximum Sample Preparation: 200 mg of sample mixed with 10 mL water sonicated for 15 min and filtered through

N 20

0 1 0 3 0 540 5 2 5 4 5

P operating pressure of 200 bar.mL water, sonicated for 15 min, and filtered through 0 2 μm PVDF membrane Flow rate F (mL/min)

0.1 0.3 0.50.5 2.5 4.5Linear Velocity u (mm/s)0.2 μm PVDF membrane

Mobile Phase A: 50 mM NH OAc pH 4 8Flow rate F (mL/min)Linear Velocity u (mm/s) ReferencesMobile Phase A: 50 mM NH4OAc, pH 4.8

Mobile Phase B: Optima LC/MS acetonitrile FIGURE 5 Peak capacities FIGURE 6: SystemReferences

1 G i h G Th Li it f th S ti P fMobile Phase B: Optima LC/MS acetonitrileG di t F 5% B t 50% B

FIGURE 5. Peak capacities (valid for all alkylphenones)

FIGURE 6: System backpressures

1. Guiochon, G. The Limits of the Separation Power of Gradient : From 5% B to 50% B, (valid for all alkylphenones). backpressures. Unidimensional Column liquid chromatography, Gradient Time: see figure (mentioned as tG)

1200J. Chromatogr. A, 2006, 1126, 6-49.

Flow Rate: see figure 220n c 1200) © 2014 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific IncInjection Volume: 2 μL ity ba

r © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe j μ

Column Temperature: 45 °C 180paci 800e(b the intellectual property rights of others.

PO71312-EN 0814SColumn Temperature: 45 CDetection wavelength: 280 nm C

ap sure

Detection wavelength: 280 nmDetection Rate: 50 Hz 140k

C

400ess

Detection Rate: 50 Hz 140

Pea 400

Pre

100

P

020 40 60 80R t ti Ti t ( i )

0 0.3 0.50.1Retention Time tR (min) Flow rate F (mL/min)

3Thermo Scientific Poster Note • PN71312-ISC-EN 0914S

Maximizing the Peak Capacity to Analysis Time Ratio in One DimensionalMaximizing the Peak-Capacity-to-Analysis-Time Ratio in One-Dimensional a g t e ea Capac ty to a ys s e at o O e e s o aCLiquid ChromatographyLiquid Chromatography

Mi h l H id S F b l d F k St iMichael Heidorn, Susanne Fabel and Frank SteinerMichael Heidorn, Susanne Fabel and Frank SteinerTh Fi h S i tifi G i GThermo Fisher Scientific, Germering, GermanyThermo Fisher Scientific, Germering, Germany

Overview Results While the theoretical plate heights (Figure 3) of both column-Overview Results p g ( g )series merged each other the same way as it was already

Purpose: A separation of complex sample mixtures with Comparison of 2 1 × 100 mm Columnsseries merged each other the same way as it was already seen in figure 1 the number of theoretical plates (Figure 4)Purpose: A separation of complex sample mixtures with

appropriate resolution in the shortest time possibleComparison of 2.1 × 100 mm Columns seen in figure 1, the number of theoretical plates (Figure 4)

was much higher for the longer sequence of Accucoreappropriate resolution in the shortest time possible. Different chromatographic columns (porous C18-silica, was much higher for the longer sequence of Accucore columns The maximum peak capacity of the AccucoreMethods: Different stationary phase architectures, column

g (fused-core silica-based C18, and monolithic silica-based columns. The maximum peak capacity of the Accucore

column chain was found to be n 220 at a retention time ofy p ,lengths, and temperatures, as well as applying pressures

,C18e) were tested by using the RP-5 standard under column chain was found to be nc = 220 at a retention time of

b t t 40 i (Fi 5) Th i k it flengths, and temperatures, as well as applying pressures up to 1200 bar

C18e) were tested by using the RP 5 standard under isocratic conditions (Figure 1 and Figure 2) about tR = 40 min (Figure 5). The maximum peak capacity of up to 1200 bar. isocratic conditions (Figure 1 and Figure 2). the Hypersil GOLD column was probably not achieved but

Results: Exploiting the maximum column pressure, the FIGURE 1: Efficiencies of columns with different found to be approximately nc = 150 at a retention time of p g p ,best peak-capacity-to-analysis-time ratio for isocratic stationary phase architectures but the same

y cabout tR = 25 min (Figure 5). The flow-pressure range was p p y y

conditions was determined by Thermo Scientific™y p

dimension of 2.1 × 100 mm.R ( g ) p g

similar for both column-series (Figure 6).conditions was determined by Thermo ScientificAccucore™ columns 2 6 μm particle size With gradient

dimension of 2.1 100 mm.Poro s Particles d 2 5 m C18 Comp Wm

)

similar for both column series (Figure 6).Accucore columns, 2.6 μm particle size. With gradient conditions Thermo Scientific™ Hypersil GOLD™ columns

Porous Particles, dp= 2.5 μm, C18, Comp. WPorous Particles d = 1 9 μm C18 Hypersil GOLD19(µ

m Traditional Chinese Medicine (TCM)conditions, Thermo Scientific Hypersil GOLD columns, 1 9 μm particle size were found to be superior

Porous Particles, dp= 1.9 μm, C18, Hypersil GOLDSolid Core Particles, dp= 2.6 μm, C18, Accucore17H

(

The series of Hypersil GOLD (2 1 × 300 mm) columns and1.9 μm particle size, were found to be superior. Solid Core Particles, dp 2.6 μm, C18, AccucoreSolid Core Particles, dp= 2.7 μm, C18, Comp. A15

17

ght The series of Hypersil GOLD (2.1 300 mm) columns and

Accucore (2 1 × 450 mm) columns were tested by using the

I t d tip μ p

Monolithic Rod, C18(e), Comp. M1315

eig

ne

Accucore (2.1 × 450 mm) columns were tested by using the TCM sample “Dang Gui Long Hui Wan“ under gradientIntroduction 13

e H

eyl

en TCM-sample Dang Gui Long Hui Wan under gradient conditions Applying the optimum linear velocity to each the

Th ti f ll l d ith i t11la

teo-

Xy conditions. Applying the optimum linear velocity to each, the th A l i i t d k itThe separation of all sample compounds with appropriate 9lP

lof

o three Accucore columns in series generated a peak capacity resolution in the shortest time possible is a common goal 7ic

al o of nc = 670 at the gradient time tg = 60 min with a of most chromatographers. This goal becomes even more 5re

ti gbackpressure of 850 bar. The two Hypersil GOLD columns in g g

relevant with highly complex samples. Generally, highest 35

eor

series generated a peak capacity of nc = 480 at the gradient g y p p y, gpeak capacities are achieved by employing an additional

30.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5Th

e g p p y c gtime tg = 28 min with a backpressure of 1200 bar.peak capacities are achieved by employing an additional

chromatographic dimension Dedicated instrumentation Linear Velocity u (mm/s)time tg 28 min with a backpressure of 1200 bar.

chromatographic dimension. Dedicated instrumentation, extensive method development and a proper combination FIGURE 2: Backpressures of columns with different FIGURE 7: Calculated peak capacities with columnextensive method development, and a proper combination of separation chemistries are necessary in this case The

FIGURE 2: Backpressures of columns with different t ti h hit t b t th

FIGURE 7: Calculated peak capacities with column chains in gradient mode: Hypersil GOLD 2 1 × 300 mmof separation chemistries are necessary in this case. The

theoretical basis has been summarized by Guiochon 1stationary phase architectures but the same chains in gradient mode: Hypersil GOLD 2.1 × 300 mm

l A 2 1 × 450 ltheoretical basis has been summarized by Guiochon.1 dimension of 2.1 × 100 mm. column or Accucore 2.1 × 450 mm column.

Highest resolution in shortest time can also be achieved Porous Particles d = 2 5 μm1000 700Highest resolution in shortest time can also be achieved by maximizing the peak-capacity-to-analysis-time ratio of

Porous Particles, dp 2.5 μmPorous Particles, dp= 1.9 μm F 0 45 0 65 0 60 0 45600

700

yby maximizing the peak-capacity-to-analysis-time ratio of an application This approach is easily implemented by

, p μSolid Core Particles, dp= 2.7 μm800) (mL/min)

0.45 0.65 0.60 0.45500600

city

an application. This approach is easily implemented by applying columns of extended length packed with porous

pSolid Core Particles, dp= 2.6 μmM lithi R dba

r)

tG 40 42 28 60500pa

c

applying columns of extended length packed with porous b 2 i ti l O th th h d h t

Monolithic Rod600e(b tG

(min)40 42 28 60400C

a

sub-2-micron particles. On the other hand, such a setup ure ( )

Pak

lead to a column head pressure up to 1200 bar and even 400ess P

(bar)930 1200 1200 850200P

e

more. In this work, experimental results from the use of Max Pressure of MonolithPr

e (bar)

n 430 600 480 670different stationary phase architectures in column lengths Max. Pressure of Monolith200

P nc 430 600 480 6700y p g

of 100 mm to 450 mm and at pressures up to 1200 bar are0

of 100 mm to 450 mm and at pressures up to 1200 bar are discussed Furthermore it is evaluated which approach 0 FIGURE 8: Ratio of peak-capacity-to-analysis-time with discussed. Furthermore, it is evaluated which approach provides the best peak-capacity-to-analysis-time ratio for

0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05Fl t F ( L/ i )

p p y ycolumn chains in gradient mode: Hypersil GOLD 2.1 ×provides the best peak-capacity-to-analysis-time ratio for

the analysis of complex sample mixturesFlow rate F (mL/min) g yp

300 mm column or Accucore 2.1 × 450 mm column.the analysis of complex sample mixtures. In one dimensional liquid chromatography the best peak

300 mm column or Accucore 2.1 450 mm column.17

M th dIn one-dimensional liquid chromatography, the best peak-

it t l i ti ti i hi d b l iti17

o FMethods capacity-to-analysis-time ratio is achieved by exploiting 14to

Rat

io F (mL/min)

0.45 0.65 0.60 0.45

Liquid Chromatography System the flow-pressure footprint of the system and column 11ci

ty

e R (mL/min)

/tLiquid Chromatography System™ ™ ™

hardware. High peak capacity is basically achieved 11

pac

Tim

e nc/tG( / )

11 14 17 11Thermo Scientific ™ Dionex™ UltiMate™ 3000 XRS system: through a high number of theoretical plates (N) per C

ap s T (1/min)

solvent rack SR-3000, pump LPG-3400XRS, sampler g g p ( ) p

column. The optimum linear velocity is determined by the 5ak C

lysi P 930 1200 1200 850p p p

WPS-3000TXRS, column thermostat TCC-3000RS, column. The optimum linear velocity is determined by the minimum theoretical plate height (Figure 1) Extending the P

eaA

nal

(bar)930 1200 1200 850

, ,and detector DAD-3000RS.

minimum theoretical plate height (Figure 1). Extending the column length means enhancing the column efficiency (N) 0

Aand detector DAD 3000RS. column length means enhancing the column efficiency (N) but also requires a higher pressure to achieve the same

0

Data Analysis but also requires a higher pressure to achieve the same linear velocity (Figure 2) The Accucore column chain provided higher peak capacities

Thermo Scientific™ Dionex™ Chromeleon™linear velocity (Figure 2). The Accucore column chain provided higher peak capacities

th th H il GOLD l h i t h fl tThermo Scientific Dionex ChromeleonChromatograph Data S stem Soft are ersion 6 8 Different concepts of stationary phase design were than the Hypersil GOLD column chain at each flow rate Chromatography Data System Software version 6.8 p y p g

compared by using identical column dimensions. The (Figure 7). The ratio between the peak capacities of both Comparison of 2 1x100 mm columns

p y gmonolithic rod showed the poorest efficiency but also column chains was the same as found under isocratic Comparison of 2.1x100 mm columns

C l Diff t d i f t ti h ( llmonolithic rod showed the poorest efficiency but also required the lowest flow-pressure footprint The maximum conditions by using phenones (Figure 5). But when

Columns: Different designs of stationary phase (all required the lowest flow-pressure footprint. The maximum operation pressure of a monolithic rod is about 200 bar

y g p ( g )comparing the ratio of peak capacity to analysis time, the

RP-C18) with 2.1 × 100 mm each operation pressure of a monolithic rod is about 200 bar and was almost reached for the 2 1 x 100 mm column at a

comparing the ratio of peak capacity to analysis time, the Accucore column chain generated 14 peaks per minute while

RP-5 Standard: Theophylline, p-nitroaniline, phenetole, and was almost reached for the 2.1 x 100 mm column at a fl t f 1 L/ i (Fi 2) H li

Accucore column chain generated 14 peaks per minute while the Hypersil GOLD column chain generated 17 peak per

o-xylene, and methyl benzoate flow rate of 1 mL/min (Figure 2). Hence, coupling li hi d h i hi h ld h l

the Hypersil GOLD column chain generated 17 peak per minute at 1200 bar backpressure (Figure 8) Thus byy , y

Mobile Phase A: Fisher Scientific™ Optima™ LC/MS water monolithic rods to a chain which would show an equal minute at 1200 bar backpressure (Figure 8). Thus, by exhausting the maximum pressure the Accucore chain wasMobile Phase A: Fisher Scientific Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrile number of theoretical plates compared to the other column exhausting the maximum pressure, the Accucore chain was bl t hi th hi h t k it b t th H ilMobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% B types is not feasible. able to achieve the highest peak capacity but the Hypersil OIsocratic Conditions: 50% A and 50% B

Flow Rate: 0 1 1 0 mL/minypHypersil GOLD and Accucore columns were found to

GOLD column chain provided the maximum ratio of peak Flow Rate: 0.1–1.0 mL/minInjection Volume:1 µL

Hypersil GOLD and Accucore columns were found to provide the highest column efficiency (Figure 1)

capacity to analysis time.Injection Volume:1 µLC l T t 30 °C

provide the highest column efficiency (Figure 1). F th th A l h d b t 60% fColumn Temperature: 30 °C Furthermore, the Accucore columns showed about 60% of th b k l ti t th H il GOLD l ConclusionDetection wavelength: 254 nm the backpressure relative to the Hypersil GOLD columns Conclusion

Detection Rate: 20 Hz at identical flow rates (Figure 2). Accordingly, to exploit the By exploiting the maximum pressure of the column Comparison of columns coupled in series flow-pressure footprint of the system and column

y p g phardware, a 2.1 × 450 mm long chain of Accucore C18Comparison of columns coupled in series

C l S i H il GOLD C18

p p yhardware, three Accucore 2.1 × 150 mm columns were

hardware, a 2.1 450 mm long chain of Accucore C18 (2 6 μm) columns could be built It produced a higherColumn-Series: Hypersil GOLD C18, ,

coupled in series to a sequence of 2.1 × 450 mm. For(2.6 μm) columns could be built. It produced a higher efficiency (N) than the 2 1 × 300 mm long Hypersil GOLD1.9 μm, 2.1 × 200 and 2.1 × 100 mm coupled in series to a sequence of 2.1 450 mm. For

Hypersil GOLD columns 2 1 × 200 mm and 2 1 × 100efficiency (N) than the 2.1 × 300 mm long Hypersil GOLD (1 9 μm) column chainAccucore C18, Hypersil GOLD columns , 2.1 × 200 mm and 2.1 × 100

mm were coupled to a sequence of 2 1 × 300 mm(1.9 μm) column chain.

2.6 μm, 2.1 × 150 mm (three) mm were coupled to a sequence of 2.1 × 300 mm. On the Accucore 2.1 × 450 mm column chain, a peakμ , ( )

Sample: Phenones and uracil Comparison of Columns Coupled in SeriesOn the Accucore 2.1 450 mm column chain, a peak capacity of 600 was achieved for the TCM “Dang GuiSample: Phenones and uracil

Mobile Phase A: Optima LC/MS waterp p

The series of Hypersil GOLD (2 1 × 300 mm) andcapacity of 600 was achieved for the TCM Dang Gui Long Hui Wan” with 42 min gradient time (14 peaks perMobile Phase A: Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrileThe series of Hypersil GOLD (2.1 × 300 mm) and Acc core (2 1 × 450 mm) col mns ere characteri ed

Long Hui Wan with 42 min gradient time (14 peaks per minute) A peak capacity of 670 could be realized with 60Mobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% BAccucore (2.1 × 450 mm) columns were characterized

ith lk l h t d d d i ti ditiminute). A peak capacity of 670 could be realized with 60

i di t ti hi h i d th k itIsocratic Conditions: 50% A and 50% BFlow Rate: 0 05 0 5 mL/min

with alkylphenone standard under isocratic conditions. min gradient time, which compromised the peak-capacity-t l i ti ti (11 k i t )Flow Rate: 0.05–0.5 mL/min

I j ti V l 5 L FIGURE 4: TheoreticalFIGURE 3 Theoretical to-analysis-time ratio (11 peaks per minute).Injection Volume: 5 μL FIGURE 4: Theoretical

plate numbers (N) ofFIGURE 3. Theoretical plate heights (H) of On the Hypersil GOLD 2 1 × 300 mm column chain aColumn Temperature: 35 °C plate numbers (N) of

hexanophenoneplate heights (H) of hexanophenone

On the Hypersil GOLD 2.1 × 300 mm column chain, a peak capacity of 430 was achieved for the TCM “DangDetection: 250 nm hexanophenone.hexanophenone. peak capacity of 430 was achieved for the TCM Dang G i L H i W “ i 40 i di t ti (11 kDetection Rate: 20 Hz N 100 x10³20m

)

Hypersil GOLD 2 1 x 300 mmGui Long Hui Wan“ in 40 min gradient time (11 peaks per

i t ) A k it f 480 d t i d ith 28T diti l Chi M di i (TCM) es 80

20

(µm

A 2 1 450

Hypersil GOLD 2.1 x 300 mm minute). A peak capacity of 480 was determined with 28 Traditional Chinese Medicine (TCM)

plat 80

16H ( Accucore 2.1 x 450 mm min gradient time, which benefited the peak-capacity-to-

Columns: see above

ofp

6016

ght analysis-time ratio (17 peaks per minute).

TCM-Sample: Dang Gui Long Hui Wan Nr. 3560 B, ero

4010Hei

g y ( p p )

M lithi l t id hi h k itiHerba Natura Harald Noll BV, Dinxperlo, the Netherlands mbe 4010

e H Monolithic columns cannot provide high peak capacities

i bl d h t ti d t th i i, p ,

Sample Preparation: 200 mg of sample mixed with 10 Num 204la

te in a comparable and short time due to their maximum Sample Preparation: 200 mg of sample mixed with 10 mL water sonicated for 15 min and filtered through

N 20

0 1 0 3 0 540 5 2 5 4 5

P operating pressure of 200 bar.mL water, sonicated for 15 min, and filtered through 0 2 μm PVDF membrane Flow rate F (mL/min)

0.1 0.3 0.50.5 2.5 4.5Linear Velocity u (mm/s)0.2 μm PVDF membrane

Mobile Phase A: 50 mM NH OAc pH 4 8Flow rate F (mL/min)Linear Velocity u (mm/s) ReferencesMobile Phase A: 50 mM NH4OAc, pH 4.8

Mobile Phase B: Optima LC/MS acetonitrile FIGURE 5 Peak capacities FIGURE 6: SystemReferences

1 G i h G Th Li it f th S ti P fMobile Phase B: Optima LC/MS acetonitrileG di t F 5% B t 50% B

FIGURE 5. Peak capacities (valid for all alkylphenones)

FIGURE 6: System backpressures

1. Guiochon, G. The Limits of the Separation Power of Gradient : From 5% B to 50% B, (valid for all alkylphenones). backpressures. Unidimensional Column liquid chromatography, Gradient Time: see figure (mentioned as tG)

1200J. Chromatogr. A, 2006, 1126, 6-49.

Flow Rate: see figure 220n c 1200) © 2014 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific IncInjection Volume: 2 μL ity ba

r © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe j μ

Column Temperature: 45 °C 180paci 800e(b the intellectual property rights of others.

PO71312-EN 0814SColumn Temperature: 45 CDetection wavelength: 280 nm C

ap sure

Detection wavelength: 280 nmDetection Rate: 50 Hz 140k

C

400ess

Detection Rate: 50 Hz 140

Pea 400

Pre

100

P

020 40 60 80R t ti Ti t ( i )

0 0.3 0.50.1Retention Time tR (min) Flow rate F (mL/min)

4 Maximizing the Peak-Capacity-to-Analysis-Time Ratio in One-Dimensional Liquid Chromatography

Maximizing the Peak Capacity to Analysis Time Ratio in One DimensionalMaximizing the Peak-Capacity-to-Analysis-Time Ratio in One-Dimensional a g t e ea Capac ty to a ys s e at o O e e s o aCLiquid ChromatographyLiquid Chromatography

Mi h l H id S F b l d F k St iMichael Heidorn, Susanne Fabel and Frank SteinerMichael Heidorn, Susanne Fabel and Frank SteinerTh Fi h S i tifi G i GThermo Fisher Scientific, Germering, GermanyThermo Fisher Scientific, Germering, Germany

Overview Results While the theoretical plate heights (Figure 3) of both column-Overview Results p g ( g )series merged each other the same way as it was already

Purpose: A separation of complex sample mixtures with Comparison of 2 1 × 100 mm Columnsseries merged each other the same way as it was already seen in figure 1 the number of theoretical plates (Figure 4)Purpose: A separation of complex sample mixtures with

appropriate resolution in the shortest time possibleComparison of 2.1 × 100 mm Columns seen in figure 1, the number of theoretical plates (Figure 4)

was much higher for the longer sequence of Accucoreappropriate resolution in the shortest time possible. Different chromatographic columns (porous C18-silica, was much higher for the longer sequence of Accucore columns The maximum peak capacity of the AccucoreMethods: Different stationary phase architectures, column

g (fused-core silica-based C18, and monolithic silica-based columns. The maximum peak capacity of the Accucore

column chain was found to be n 220 at a retention time ofy p ,lengths, and temperatures, as well as applying pressures

,C18e) were tested by using the RP-5 standard under column chain was found to be nc = 220 at a retention time of

b t t 40 i (Fi 5) Th i k it flengths, and temperatures, as well as applying pressures up to 1200 bar

C18e) were tested by using the RP 5 standard under isocratic conditions (Figure 1 and Figure 2) about tR = 40 min (Figure 5). The maximum peak capacity of up to 1200 bar. isocratic conditions (Figure 1 and Figure 2). the Hypersil GOLD column was probably not achieved but

Results: Exploiting the maximum column pressure, the FIGURE 1: Efficiencies of columns with different found to be approximately nc = 150 at a retention time of p g p ,best peak-capacity-to-analysis-time ratio for isocratic stationary phase architectures but the same

y cabout tR = 25 min (Figure 5). The flow-pressure range was p p y y

conditions was determined by Thermo Scientific™y p

dimension of 2.1 × 100 mm.R ( g ) p g

similar for both column-series (Figure 6).conditions was determined by Thermo ScientificAccucore™ columns 2 6 μm particle size With gradient

dimension of 2.1 100 mm.Poro s Particles d 2 5 m C18 Comp Wm

)

similar for both column series (Figure 6).Accucore columns, 2.6 μm particle size. With gradient conditions Thermo Scientific™ Hypersil GOLD™ columns

Porous Particles, dp= 2.5 μm, C18, Comp. WPorous Particles d = 1 9 μm C18 Hypersil GOLD19(µ

m Traditional Chinese Medicine (TCM)conditions, Thermo Scientific Hypersil GOLD columns, 1 9 μm particle size were found to be superior

Porous Particles, dp= 1.9 μm, C18, Hypersil GOLDSolid Core Particles, dp= 2.6 μm, C18, Accucore17H

(

The series of Hypersil GOLD (2 1 × 300 mm) columns and1.9 μm particle size, were found to be superior. Solid Core Particles, dp 2.6 μm, C18, AccucoreSolid Core Particles, dp= 2.7 μm, C18, Comp. A15

17

ght The series of Hypersil GOLD (2.1 300 mm) columns and

Accucore (2 1 × 450 mm) columns were tested by using the

I t d tip μ p

Monolithic Rod, C18(e), Comp. M1315

eig

ne

Accucore (2.1 × 450 mm) columns were tested by using the TCM sample “Dang Gui Long Hui Wan“ under gradientIntroduction 13

e H

eyl

en TCM-sample Dang Gui Long Hui Wan under gradient conditions Applying the optimum linear velocity to each the

Th ti f ll l d ith i t11la

teo-

Xy conditions. Applying the optimum linear velocity to each, the th A l i i t d k itThe separation of all sample compounds with appropriate 9lP

lof

o three Accucore columns in series generated a peak capacity resolution in the shortest time possible is a common goal 7ic

al o of nc = 670 at the gradient time tg = 60 min with a of most chromatographers. This goal becomes even more 5re

ti gbackpressure of 850 bar. The two Hypersil GOLD columns in g g

relevant with highly complex samples. Generally, highest 35

eor

series generated a peak capacity of nc = 480 at the gradient g y p p y, gpeak capacities are achieved by employing an additional

30.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5Th

e g p p y c gtime tg = 28 min with a backpressure of 1200 bar.peak capacities are achieved by employing an additional

chromatographic dimension Dedicated instrumentation Linear Velocity u (mm/s)time tg 28 min with a backpressure of 1200 bar.

chromatographic dimension. Dedicated instrumentation, extensive method development and a proper combination FIGURE 2: Backpressures of columns with different FIGURE 7: Calculated peak capacities with columnextensive method development, and a proper combination of separation chemistries are necessary in this case The

FIGURE 2: Backpressures of columns with different t ti h hit t b t th

FIGURE 7: Calculated peak capacities with column chains in gradient mode: Hypersil GOLD 2 1 × 300 mmof separation chemistries are necessary in this case. The

theoretical basis has been summarized by Guiochon 1stationary phase architectures but the same chains in gradient mode: Hypersil GOLD 2.1 × 300 mm

l A 2 1 × 450 ltheoretical basis has been summarized by Guiochon.1 dimension of 2.1 × 100 mm. column or Accucore 2.1 × 450 mm column.

Highest resolution in shortest time can also be achieved Porous Particles d = 2 5 μm1000 700Highest resolution in shortest time can also be achieved by maximizing the peak-capacity-to-analysis-time ratio of

Porous Particles, dp 2.5 μmPorous Particles, dp= 1.9 μm F 0 45 0 65 0 60 0 45600

700

yby maximizing the peak-capacity-to-analysis-time ratio of an application This approach is easily implemented by

, p μSolid Core Particles, dp= 2.7 μm800) (mL/min)

0.45 0.65 0.60 0.45500600

city

an application. This approach is easily implemented by applying columns of extended length packed with porous

pSolid Core Particles, dp= 2.6 μmM lithi R dba

r)

tG 40 42 28 60500pa

c

applying columns of extended length packed with porous b 2 i ti l O th th h d h t

Monolithic Rod600e(b tG

(min)40 42 28 60400C

a

sub-2-micron particles. On the other hand, such a setup ure ( )

Pak

lead to a column head pressure up to 1200 bar and even 400ess P

(bar)930 1200 1200 850200P

e

more. In this work, experimental results from the use of Max Pressure of MonolithPr

e (bar)

n 430 600 480 670different stationary phase architectures in column lengths Max. Pressure of Monolith200

P nc 430 600 480 6700y p g

of 100 mm to 450 mm and at pressures up to 1200 bar are0

of 100 mm to 450 mm and at pressures up to 1200 bar are discussed Furthermore it is evaluated which approach 0 FIGURE 8: Ratio of peak-capacity-to-analysis-time with discussed. Furthermore, it is evaluated which approach provides the best peak-capacity-to-analysis-time ratio for

0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05Fl t F ( L/ i )

p p y ycolumn chains in gradient mode: Hypersil GOLD 2.1 ×provides the best peak-capacity-to-analysis-time ratio for

the analysis of complex sample mixturesFlow rate F (mL/min) g yp

300 mm column or Accucore 2.1 × 450 mm column.the analysis of complex sample mixtures. In one dimensional liquid chromatography the best peak

300 mm column or Accucore 2.1 450 mm column.17

M th dIn one-dimensional liquid chromatography, the best peak-

it t l i ti ti i hi d b l iti17

o FMethods capacity-to-analysis-time ratio is achieved by exploiting 14to

Rat

io F (mL/min)

0.45 0.65 0.60 0.45

Liquid Chromatography System the flow-pressure footprint of the system and column 11ci

ty

e R (mL/min)

/tLiquid Chromatography System™ ™ ™

hardware. High peak capacity is basically achieved 11

pac

Tim

e nc/tG( / )

11 14 17 11Thermo Scientific ™ Dionex™ UltiMate™ 3000 XRS system: through a high number of theoretical plates (N) per C

ap s T (1/min)

solvent rack SR-3000, pump LPG-3400XRS, sampler g g p ( ) p

column. The optimum linear velocity is determined by the 5ak C

lysi P 930 1200 1200 850p p p

WPS-3000TXRS, column thermostat TCC-3000RS, column. The optimum linear velocity is determined by the minimum theoretical plate height (Figure 1) Extending the P

eaA

nal

(bar)930 1200 1200 850

, ,and detector DAD-3000RS.

minimum theoretical plate height (Figure 1). Extending the column length means enhancing the column efficiency (N) 0

Aand detector DAD 3000RS. column length means enhancing the column efficiency (N) but also requires a higher pressure to achieve the same

0

Data Analysis but also requires a higher pressure to achieve the same linear velocity (Figure 2) The Accucore column chain provided higher peak capacities

Thermo Scientific™ Dionex™ Chromeleon™linear velocity (Figure 2). The Accucore column chain provided higher peak capacities

th th H il GOLD l h i t h fl tThermo Scientific Dionex ChromeleonChromatograph Data S stem Soft are ersion 6 8 Different concepts of stationary phase design were than the Hypersil GOLD column chain at each flow rate Chromatography Data System Software version 6.8 p y p g

compared by using identical column dimensions. The (Figure 7). The ratio between the peak capacities of both Comparison of 2 1x100 mm columns

p y gmonolithic rod showed the poorest efficiency but also column chains was the same as found under isocratic Comparison of 2.1x100 mm columns

C l Diff t d i f t ti h ( llmonolithic rod showed the poorest efficiency but also required the lowest flow-pressure footprint The maximum conditions by using phenones (Figure 5). But when

Columns: Different designs of stationary phase (all required the lowest flow-pressure footprint. The maximum operation pressure of a monolithic rod is about 200 bar

y g p ( g )comparing the ratio of peak capacity to analysis time, the

RP-C18) with 2.1 × 100 mm each operation pressure of a monolithic rod is about 200 bar and was almost reached for the 2 1 x 100 mm column at a

comparing the ratio of peak capacity to analysis time, the Accucore column chain generated 14 peaks per minute while

RP-5 Standard: Theophylline, p-nitroaniline, phenetole, and was almost reached for the 2.1 x 100 mm column at a fl t f 1 L/ i (Fi 2) H li

Accucore column chain generated 14 peaks per minute while the Hypersil GOLD column chain generated 17 peak per

o-xylene, and methyl benzoate flow rate of 1 mL/min (Figure 2). Hence, coupling li hi d h i hi h ld h l

the Hypersil GOLD column chain generated 17 peak per minute at 1200 bar backpressure (Figure 8) Thus byy , y

Mobile Phase A: Fisher Scientific™ Optima™ LC/MS water monolithic rods to a chain which would show an equal minute at 1200 bar backpressure (Figure 8). Thus, by exhausting the maximum pressure the Accucore chain wasMobile Phase A: Fisher Scientific Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrile number of theoretical plates compared to the other column exhausting the maximum pressure, the Accucore chain was bl t hi th hi h t k it b t th H ilMobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% B types is not feasible. able to achieve the highest peak capacity but the Hypersil OIsocratic Conditions: 50% A and 50% B

Flow Rate: 0 1 1 0 mL/minypHypersil GOLD and Accucore columns were found to

GOLD column chain provided the maximum ratio of peak Flow Rate: 0.1–1.0 mL/minInjection Volume:1 µL

Hypersil GOLD and Accucore columns were found to provide the highest column efficiency (Figure 1)

capacity to analysis time.Injection Volume:1 µLC l T t 30 °C

provide the highest column efficiency (Figure 1). F th th A l h d b t 60% fColumn Temperature: 30 °C Furthermore, the Accucore columns showed about 60% of th b k l ti t th H il GOLD l ConclusionDetection wavelength: 254 nm the backpressure relative to the Hypersil GOLD columns Conclusion

Detection Rate: 20 Hz at identical flow rates (Figure 2). Accordingly, to exploit the By exploiting the maximum pressure of the column Comparison of columns coupled in series flow-pressure footprint of the system and column

y p g phardware, a 2.1 × 450 mm long chain of Accucore C18Comparison of columns coupled in series

C l S i H il GOLD C18

p p yhardware, three Accucore 2.1 × 150 mm columns were

hardware, a 2.1 450 mm long chain of Accucore C18 (2 6 μm) columns could be built It produced a higherColumn-Series: Hypersil GOLD C18, ,

coupled in series to a sequence of 2.1 × 450 mm. For(2.6 μm) columns could be built. It produced a higher efficiency (N) than the 2 1 × 300 mm long Hypersil GOLD1.9 μm, 2.1 × 200 and 2.1 × 100 mm coupled in series to a sequence of 2.1 450 mm. For

Hypersil GOLD columns 2 1 × 200 mm and 2 1 × 100efficiency (N) than the 2.1 × 300 mm long Hypersil GOLD (1 9 μm) column chainAccucore C18, Hypersil GOLD columns , 2.1 × 200 mm and 2.1 × 100

mm were coupled to a sequence of 2 1 × 300 mm(1.9 μm) column chain.

2.6 μm, 2.1 × 150 mm (three) mm were coupled to a sequence of 2.1 × 300 mm. On the Accucore 2.1 × 450 mm column chain, a peakμ , ( )

Sample: Phenones and uracil Comparison of Columns Coupled in SeriesOn the Accucore 2.1 450 mm column chain, a peak capacity of 600 was achieved for the TCM “Dang GuiSample: Phenones and uracil

Mobile Phase A: Optima LC/MS waterp p

The series of Hypersil GOLD (2 1 × 300 mm) andcapacity of 600 was achieved for the TCM Dang Gui Long Hui Wan” with 42 min gradient time (14 peaks perMobile Phase A: Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrileThe series of Hypersil GOLD (2.1 × 300 mm) and Acc core (2 1 × 450 mm) col mns ere characteri ed

Long Hui Wan with 42 min gradient time (14 peaks per minute) A peak capacity of 670 could be realized with 60Mobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% BAccucore (2.1 × 450 mm) columns were characterized

ith lk l h t d d d i ti ditiminute). A peak capacity of 670 could be realized with 60

i di t ti hi h i d th k itIsocratic Conditions: 50% A and 50% BFlow Rate: 0 05 0 5 mL/min

with alkylphenone standard under isocratic conditions. min gradient time, which compromised the peak-capacity-t l i ti ti (11 k i t )Flow Rate: 0.05–0.5 mL/min

I j ti V l 5 L FIGURE 4: TheoreticalFIGURE 3 Theoretical to-analysis-time ratio (11 peaks per minute).Injection Volume: 5 μL FIGURE 4: Theoretical

plate numbers (N) ofFIGURE 3. Theoretical plate heights (H) of On the Hypersil GOLD 2 1 × 300 mm column chain aColumn Temperature: 35 °C plate numbers (N) of

hexanophenoneplate heights (H) of hexanophenone

On the Hypersil GOLD 2.1 × 300 mm column chain, a peak capacity of 430 was achieved for the TCM “DangDetection: 250 nm hexanophenone.hexanophenone. peak capacity of 430 was achieved for the TCM Dang G i L H i W “ i 40 i di t ti (11 kDetection Rate: 20 Hz N 100 x10³20m

)

Hypersil GOLD 2 1 x 300 mmGui Long Hui Wan“ in 40 min gradient time (11 peaks per

i t ) A k it f 480 d t i d ith 28T diti l Chi M di i (TCM) es 80

20

(µm

A 2 1 450

Hypersil GOLD 2.1 x 300 mm minute). A peak capacity of 480 was determined with 28 Traditional Chinese Medicine (TCM)

plat 80

16H ( Accucore 2.1 x 450 mm min gradient time, which benefited the peak-capacity-to-

Columns: see above

ofp

6016

ght analysis-time ratio (17 peaks per minute).

TCM-Sample: Dang Gui Long Hui Wan Nr. 3560 B, ero

4010Hei

g y ( p p )

M lithi l t id hi h k itiHerba Natura Harald Noll BV, Dinxperlo, the Netherlands mbe 4010

e H Monolithic columns cannot provide high peak capacities

i bl d h t ti d t th i i, p ,

Sample Preparation: 200 mg of sample mixed with 10 Num 204la

te in a comparable and short time due to their maximum Sample Preparation: 200 mg of sample mixed with 10 mL water sonicated for 15 min and filtered through

N 20

0 1 0 3 0 540 5 2 5 4 5

P operating pressure of 200 bar.mL water, sonicated for 15 min, and filtered through 0 2 μm PVDF membrane Flow rate F (mL/min)

0.1 0.3 0.50.5 2.5 4.5Linear Velocity u (mm/s)0.2 μm PVDF membrane

Mobile Phase A: 50 mM NH OAc pH 4 8Flow rate F (mL/min)Linear Velocity u (mm/s) ReferencesMobile Phase A: 50 mM NH4OAc, pH 4.8

Mobile Phase B: Optima LC/MS acetonitrile FIGURE 5 Peak capacities FIGURE 6: SystemReferences

1 G i h G Th Li it f th S ti P fMobile Phase B: Optima LC/MS acetonitrileG di t F 5% B t 50% B

FIGURE 5. Peak capacities (valid for all alkylphenones)

FIGURE 6: System backpressures

1. Guiochon, G. The Limits of the Separation Power of Gradient : From 5% B to 50% B, (valid for all alkylphenones). backpressures. Unidimensional Column liquid chromatography, Gradient Time: see figure (mentioned as tG)

1200J. Chromatogr. A, 2006, 1126, 6-49.

Flow Rate: see figure 220n c 1200) © 2014 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific IncInjection Volume: 2 μL ity ba

r © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe j μ

Column Temperature: 45 °C 180paci 800e(b the intellectual property rights of others.

PO71312-EN 0814SColumn Temperature: 45 CDetection wavelength: 280 nm C

ap sure

Detection wavelength: 280 nmDetection Rate: 50 Hz 140k

C

400ess

Detection Rate: 50 Hz 140

Pea 400

Pre

100

P

020 40 60 80R t ti Ti t ( i )

0 0.3 0.50.1Retention Time tR (min) Flow rate F (mL/min)

Maximizing the Peak Capacity to Analysis Time Ratio in One DimensionalMaximizing the Peak-Capacity-to-Analysis-Time Ratio in One-Dimensional a g t e ea Capac ty to a ys s e at o O e e s o aCLiquid ChromatographyLiquid Chromatography

Mi h l H id S F b l d F k St iMichael Heidorn, Susanne Fabel and Frank SteinerMichael Heidorn, Susanne Fabel and Frank SteinerTh Fi h S i tifi G i GThermo Fisher Scientific, Germering, GermanyThermo Fisher Scientific, Germering, Germany

Overview Results While the theoretical plate heights (Figure 3) of both column-Overview Results p g ( g )series merged each other the same way as it was already

Purpose: A separation of complex sample mixtures with Comparison of 2 1 × 100 mm Columnsseries merged each other the same way as it was already seen in figure 1 the number of theoretical plates (Figure 4)Purpose: A separation of complex sample mixtures with

appropriate resolution in the shortest time possibleComparison of 2.1 × 100 mm Columns seen in figure 1, the number of theoretical plates (Figure 4)

was much higher for the longer sequence of Accucoreappropriate resolution in the shortest time possible. Different chromatographic columns (porous C18-silica, was much higher for the longer sequence of Accucore columns The maximum peak capacity of the AccucoreMethods: Different stationary phase architectures, column

g (fused-core silica-based C18, and monolithic silica-based columns. The maximum peak capacity of the Accucore

column chain was found to be n 220 at a retention time ofy p ,lengths, and temperatures, as well as applying pressures

,C18e) were tested by using the RP-5 standard under column chain was found to be nc = 220 at a retention time of

b t t 40 i (Fi 5) Th i k it flengths, and temperatures, as well as applying pressures up to 1200 bar

C18e) were tested by using the RP 5 standard under isocratic conditions (Figure 1 and Figure 2) about tR = 40 min (Figure 5). The maximum peak capacity of up to 1200 bar. isocratic conditions (Figure 1 and Figure 2). the Hypersil GOLD column was probably not achieved but

Results: Exploiting the maximum column pressure, the FIGURE 1: Efficiencies of columns with different found to be approximately nc = 150 at a retention time of p g p ,best peak-capacity-to-analysis-time ratio for isocratic stationary phase architectures but the same

y cabout tR = 25 min (Figure 5). The flow-pressure range was p p y y

conditions was determined by Thermo Scientific™y p

dimension of 2.1 × 100 mm.R ( g ) p g

similar for both column-series (Figure 6).conditions was determined by Thermo ScientificAccucore™ columns 2 6 μm particle size With gradient

dimension of 2.1 100 mm.Poro s Particles d 2 5 m C18 Comp Wm

)

similar for both column series (Figure 6).Accucore columns, 2.6 μm particle size. With gradient conditions Thermo Scientific™ Hypersil GOLD™ columns

Porous Particles, dp= 2.5 μm, C18, Comp. WPorous Particles d = 1 9 μm C18 Hypersil GOLD19(µ

m Traditional Chinese Medicine (TCM)conditions, Thermo Scientific Hypersil GOLD columns, 1 9 μm particle size were found to be superior

Porous Particles, dp= 1.9 μm, C18, Hypersil GOLDSolid Core Particles, dp= 2.6 μm, C18, Accucore17H

(

The series of Hypersil GOLD (2 1 × 300 mm) columns and1.9 μm particle size, were found to be superior. Solid Core Particles, dp 2.6 μm, C18, AccucoreSolid Core Particles, dp= 2.7 μm, C18, Comp. A15

17

ght The series of Hypersil GOLD (2.1 300 mm) columns and

Accucore (2 1 × 450 mm) columns were tested by using the

I t d tip μ p

Monolithic Rod, C18(e), Comp. M1315

eig

ne

Accucore (2.1 × 450 mm) columns were tested by using the TCM sample “Dang Gui Long Hui Wan“ under gradientIntroduction 13

e H

eyl

en TCM-sample Dang Gui Long Hui Wan under gradient conditions Applying the optimum linear velocity to each the

Th ti f ll l d ith i t11la

teo-

Xy conditions. Applying the optimum linear velocity to each, the th A l i i t d k itThe separation of all sample compounds with appropriate 9lP

lof

o three Accucore columns in series generated a peak capacity resolution in the shortest time possible is a common goal 7ic

al o of nc = 670 at the gradient time tg = 60 min with a of most chromatographers. This goal becomes even more 5re

ti gbackpressure of 850 bar. The two Hypersil GOLD columns in g g

relevant with highly complex samples. Generally, highest 35

eor

series generated a peak capacity of nc = 480 at the gradient g y p p y, gpeak capacities are achieved by employing an additional

30.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5Th

e g p p y c gtime tg = 28 min with a backpressure of 1200 bar.peak capacities are achieved by employing an additional

chromatographic dimension Dedicated instrumentation Linear Velocity u (mm/s)time tg 28 min with a backpressure of 1200 bar.

chromatographic dimension. Dedicated instrumentation, extensive method development and a proper combination FIGURE 2: Backpressures of columns with different FIGURE 7: Calculated peak capacities with columnextensive method development, and a proper combination of separation chemistries are necessary in this case The

FIGURE 2: Backpressures of columns with different t ti h hit t b t th

FIGURE 7: Calculated peak capacities with column chains in gradient mode: Hypersil GOLD 2 1 × 300 mmof separation chemistries are necessary in this case. The

theoretical basis has been summarized by Guiochon 1stationary phase architectures but the same chains in gradient mode: Hypersil GOLD 2.1 × 300 mm

l A 2 1 × 450 ltheoretical basis has been summarized by Guiochon.1 dimension of 2.1 × 100 mm. column or Accucore 2.1 × 450 mm column.

Highest resolution in shortest time can also be achieved Porous Particles d = 2 5 μm1000 700Highest resolution in shortest time can also be achieved by maximizing the peak-capacity-to-analysis-time ratio of

Porous Particles, dp 2.5 μmPorous Particles, dp= 1.9 μm F 0 45 0 65 0 60 0 45600

700

yby maximizing the peak-capacity-to-analysis-time ratio of an application This approach is easily implemented by

, p μSolid Core Particles, dp= 2.7 μm800) (mL/min)

0.45 0.65 0.60 0.45500600

city

an application. This approach is easily implemented by applying columns of extended length packed with porous

pSolid Core Particles, dp= 2.6 μmM lithi R dba

r)

tG 40 42 28 60500pa

c

applying columns of extended length packed with porous b 2 i ti l O th th h d h t

Monolithic Rod600e(b tG

(min)40 42 28 60400C

a

sub-2-micron particles. On the other hand, such a setup ure ( )

Pak

lead to a column head pressure up to 1200 bar and even 400ess P

(bar)930 1200 1200 850200P

e

more. In this work, experimental results from the use of Max Pressure of MonolithPr

e (bar)

n 430 600 480 670different stationary phase architectures in column lengths Max. Pressure of Monolith200

P nc 430 600 480 6700y p g

of 100 mm to 450 mm and at pressures up to 1200 bar are0

of 100 mm to 450 mm and at pressures up to 1200 bar are discussed Furthermore it is evaluated which approach 0 FIGURE 8: Ratio of peak-capacity-to-analysis-time with discussed. Furthermore, it is evaluated which approach provides the best peak-capacity-to-analysis-time ratio for

0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05Fl t F ( L/ i )

p p y ycolumn chains in gradient mode: Hypersil GOLD 2.1 ×provides the best peak-capacity-to-analysis-time ratio for

the analysis of complex sample mixturesFlow rate F (mL/min) g yp

300 mm column or Accucore 2.1 × 450 mm column.the analysis of complex sample mixtures. In one dimensional liquid chromatography the best peak

300 mm column or Accucore 2.1 450 mm column.17

M th dIn one-dimensional liquid chromatography, the best peak-

it t l i ti ti i hi d b l iti17

o FMethods capacity-to-analysis-time ratio is achieved by exploiting 14to

Rat

io F (mL/min)

0.45 0.65 0.60 0.45

Liquid Chromatography System the flow-pressure footprint of the system and column 11ci

ty

e R (mL/min)

/tLiquid Chromatography System™ ™ ™

hardware. High peak capacity is basically achieved 11

pac

Tim

e nc/tG( / )

11 14 17 11Thermo Scientific ™ Dionex™ UltiMate™ 3000 XRS system: through a high number of theoretical plates (N) per C

ap s T (1/min)

solvent rack SR-3000, pump LPG-3400XRS, sampler g g p ( ) p

column. The optimum linear velocity is determined by the 5ak C

lysi P 930 1200 1200 850p p p

WPS-3000TXRS, column thermostat TCC-3000RS, column. The optimum linear velocity is determined by the minimum theoretical plate height (Figure 1) Extending the P

eaA

nal

(bar)930 1200 1200 850

, ,and detector DAD-3000RS.

minimum theoretical plate height (Figure 1). Extending the column length means enhancing the column efficiency (N) 0

Aand detector DAD 3000RS. column length means enhancing the column efficiency (N) but also requires a higher pressure to achieve the same

0

Data Analysis but also requires a higher pressure to achieve the same linear velocity (Figure 2) The Accucore column chain provided higher peak capacities

Thermo Scientific™ Dionex™ Chromeleon™linear velocity (Figure 2). The Accucore column chain provided higher peak capacities

th th H il GOLD l h i t h fl tThermo Scientific Dionex ChromeleonChromatograph Data S stem Soft are ersion 6 8 Different concepts of stationary phase design were than the Hypersil GOLD column chain at each flow rate Chromatography Data System Software version 6.8 p y p g

compared by using identical column dimensions. The (Figure 7). The ratio between the peak capacities of both Comparison of 2 1x100 mm columns

p y gmonolithic rod showed the poorest efficiency but also column chains was the same as found under isocratic Comparison of 2.1x100 mm columns

C l Diff t d i f t ti h ( llmonolithic rod showed the poorest efficiency but also required the lowest flow-pressure footprint The maximum conditions by using phenones (Figure 5). But when

Columns: Different designs of stationary phase (all required the lowest flow-pressure footprint. The maximum operation pressure of a monolithic rod is about 200 bar

y g p ( g )comparing the ratio of peak capacity to analysis time, the

RP-C18) with 2.1 × 100 mm each operation pressure of a monolithic rod is about 200 bar and was almost reached for the 2 1 x 100 mm column at a

comparing the ratio of peak capacity to analysis time, the Accucore column chain generated 14 peaks per minute while

RP-5 Standard: Theophylline, p-nitroaniline, phenetole, and was almost reached for the 2.1 x 100 mm column at a fl t f 1 L/ i (Fi 2) H li

Accucore column chain generated 14 peaks per minute while the Hypersil GOLD column chain generated 17 peak per

o-xylene, and methyl benzoate flow rate of 1 mL/min (Figure 2). Hence, coupling li hi d h i hi h ld h l

the Hypersil GOLD column chain generated 17 peak per minute at 1200 bar backpressure (Figure 8) Thus byy , y

Mobile Phase A: Fisher Scientific™ Optima™ LC/MS water monolithic rods to a chain which would show an equal minute at 1200 bar backpressure (Figure 8). Thus, by exhausting the maximum pressure the Accucore chain wasMobile Phase A: Fisher Scientific Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrile number of theoretical plates compared to the other column exhausting the maximum pressure, the Accucore chain was bl t hi th hi h t k it b t th H ilMobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% B types is not feasible. able to achieve the highest peak capacity but the Hypersil OIsocratic Conditions: 50% A and 50% B

Flow Rate: 0 1 1 0 mL/minypHypersil GOLD and Accucore columns were found to

GOLD column chain provided the maximum ratio of peak Flow Rate: 0.1–1.0 mL/minInjection Volume:1 µL

Hypersil GOLD and Accucore columns were found to provide the highest column efficiency (Figure 1)

capacity to analysis time.Injection Volume:1 µLC l T t 30 °C

provide the highest column efficiency (Figure 1). F th th A l h d b t 60% fColumn Temperature: 30 °C Furthermore, the Accucore columns showed about 60% of th b k l ti t th H il GOLD l ConclusionDetection wavelength: 254 nm the backpressure relative to the Hypersil GOLD columns Conclusion

Detection Rate: 20 Hz at identical flow rates (Figure 2). Accordingly, to exploit the By exploiting the maximum pressure of the column Comparison of columns coupled in series flow-pressure footprint of the system and column

y p g phardware, a 2.1 × 450 mm long chain of Accucore C18Comparison of columns coupled in series

C l S i H il GOLD C18

p p yhardware, three Accucore 2.1 × 150 mm columns were

hardware, a 2.1 450 mm long chain of Accucore C18 (2 6 μm) columns could be built It produced a higherColumn-Series: Hypersil GOLD C18, ,

coupled in series to a sequence of 2.1 × 450 mm. For(2.6 μm) columns could be built. It produced a higher efficiency (N) than the 2 1 × 300 mm long Hypersil GOLD1.9 μm, 2.1 × 200 and 2.1 × 100 mm coupled in series to a sequence of 2.1 450 mm. For

Hypersil GOLD columns 2 1 × 200 mm and 2 1 × 100efficiency (N) than the 2.1 × 300 mm long Hypersil GOLD (1 9 μm) column chainAccucore C18, Hypersil GOLD columns , 2.1 × 200 mm and 2.1 × 100

mm were coupled to a sequence of 2 1 × 300 mm(1.9 μm) column chain.

2.6 μm, 2.1 × 150 mm (three) mm were coupled to a sequence of 2.1 × 300 mm. On the Accucore 2.1 × 450 mm column chain, a peakμ , ( )

Sample: Phenones and uracil Comparison of Columns Coupled in SeriesOn the Accucore 2.1 450 mm column chain, a peak capacity of 600 was achieved for the TCM “Dang GuiSample: Phenones and uracil

Mobile Phase A: Optima LC/MS waterp p

The series of Hypersil GOLD (2 1 × 300 mm) andcapacity of 600 was achieved for the TCM Dang Gui Long Hui Wan” with 42 min gradient time (14 peaks perMobile Phase A: Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrileThe series of Hypersil GOLD (2.1 × 300 mm) and Acc core (2 1 × 450 mm) col mns ere characteri ed

Long Hui Wan with 42 min gradient time (14 peaks per minute) A peak capacity of 670 could be realized with 60Mobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% BAccucore (2.1 × 450 mm) columns were characterized

ith lk l h t d d d i ti ditiminute). A peak capacity of 670 could be realized with 60

i di t ti hi h i d th k itIsocratic Conditions: 50% A and 50% BFlow Rate: 0 05 0 5 mL/min

with alkylphenone standard under isocratic conditions. min gradient time, which compromised the peak-capacity-t l i ti ti (11 k i t )Flow Rate: 0.05–0.5 mL/min

I j ti V l 5 L FIGURE 4: TheoreticalFIGURE 3 Theoretical to-analysis-time ratio (11 peaks per minute).Injection Volume: 5 μL FIGURE 4: Theoretical

plate numbers (N) ofFIGURE 3. Theoretical plate heights (H) of On the Hypersil GOLD 2 1 × 300 mm column chain aColumn Temperature: 35 °C plate numbers (N) of

hexanophenoneplate heights (H) of hexanophenone

On the Hypersil GOLD 2.1 × 300 mm column chain, a peak capacity of 430 was achieved for the TCM “DangDetection: 250 nm hexanophenone.hexanophenone. peak capacity of 430 was achieved for the TCM Dang G i L H i W “ i 40 i di t ti (11 kDetection Rate: 20 Hz N 100 x10³20m

)

Hypersil GOLD 2 1 x 300 mmGui Long Hui Wan“ in 40 min gradient time (11 peaks per

i t ) A k it f 480 d t i d ith 28T diti l Chi M di i (TCM) es 80

20

(µm

A 2 1 450

Hypersil GOLD 2.1 x 300 mm minute). A peak capacity of 480 was determined with 28 Traditional Chinese Medicine (TCM)

plat 80

16H ( Accucore 2.1 x 450 mm min gradient time, which benefited the peak-capacity-to-

Columns: see above

ofp

6016

ght analysis-time ratio (17 peaks per minute).

TCM-Sample: Dang Gui Long Hui Wan Nr. 3560 B, ero

4010Hei

g y ( p p )

M lithi l t id hi h k itiHerba Natura Harald Noll BV, Dinxperlo, the Netherlands mbe 4010

e H Monolithic columns cannot provide high peak capacities

i bl d h t ti d t th i i, p ,

Sample Preparation: 200 mg of sample mixed with 10 Num 204la

te in a comparable and short time due to their maximum Sample Preparation: 200 mg of sample mixed with 10 mL water sonicated for 15 min and filtered through

N 20

0 1 0 3 0 540 5 2 5 4 5

P operating pressure of 200 bar.mL water, sonicated for 15 min, and filtered through 0 2 μm PVDF membrane Flow rate F (mL/min)

0.1 0.3 0.50.5 2.5 4.5Linear Velocity u (mm/s)0.2 μm PVDF membrane

Mobile Phase A: 50 mM NH OAc pH 4 8Flow rate F (mL/min)Linear Velocity u (mm/s) ReferencesMobile Phase A: 50 mM NH4OAc, pH 4.8

Mobile Phase B: Optima LC/MS acetonitrile FIGURE 5 Peak capacities FIGURE 6: SystemReferences

1 G i h G Th Li it f th S ti P fMobile Phase B: Optima LC/MS acetonitrileG di t F 5% B t 50% B

FIGURE 5. Peak capacities (valid for all alkylphenones)

FIGURE 6: System backpressures

1. Guiochon, G. The Limits of the Separation Power of Gradient : From 5% B to 50% B, (valid for all alkylphenones). backpressures. Unidimensional Column liquid chromatography, Gradient Time: see figure (mentioned as tG)

1200J. Chromatogr. A, 2006, 1126, 6-49.

Flow Rate: see figure 220n c 1200) © 2014 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific IncInjection Volume: 2 μL ity ba

r © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe j μ

Column Temperature: 45 °C 180paci 800e(b the intellectual property rights of others.

PO71312-EN 0814SColumn Temperature: 45 CDetection wavelength: 280 nm C

ap sure

Detection wavelength: 280 nmDetection Rate: 50 Hz 140k

C

400ess

Detection Rate: 50 Hz 140

Pea 400

Pre

100

P

020 40 60 80R t ti Ti t ( i )

0 0.3 0.50.1Retention Time tR (min) Flow rate F (mL/min)

5Thermo Scientific Poster Note • PN71312-ISC-EN 0914S

Maximizing the Peak Capacity to Analysis Time Ratio in One DimensionalMaximizing the Peak-Capacity-to-Analysis-Time Ratio in One-Dimensional a g t e ea Capac ty to a ys s e at o O e e s o aCLiquid ChromatographyLiquid Chromatography

Mi h l H id S F b l d F k St iMichael Heidorn, Susanne Fabel and Frank SteinerMichael Heidorn, Susanne Fabel and Frank SteinerTh Fi h S i tifi G i GThermo Fisher Scientific, Germering, GermanyThermo Fisher Scientific, Germering, Germany

Overview Results While the theoretical plate heights (Figure 3) of both column-Overview Results p g ( g )series merged each other the same way as it was already

Purpose: A separation of complex sample mixtures with Comparison of 2 1 × 100 mm Columnsseries merged each other the same way as it was already seen in figure 1 the number of theoretical plates (Figure 4)Purpose: A separation of complex sample mixtures with

appropriate resolution in the shortest time possibleComparison of 2.1 × 100 mm Columns seen in figure 1, the number of theoretical plates (Figure 4)

was much higher for the longer sequence of Accucoreappropriate resolution in the shortest time possible. Different chromatographic columns (porous C18-silica, was much higher for the longer sequence of Accucore columns The maximum peak capacity of the AccucoreMethods: Different stationary phase architectures, column

g (fused-core silica-based C18, and monolithic silica-based columns. The maximum peak capacity of the Accucore

column chain was found to be n 220 at a retention time ofy p ,lengths, and temperatures, as well as applying pressures

,C18e) were tested by using the RP-5 standard under column chain was found to be nc = 220 at a retention time of

b t t 40 i (Fi 5) Th i k it flengths, and temperatures, as well as applying pressures up to 1200 bar

C18e) were tested by using the RP 5 standard under isocratic conditions (Figure 1 and Figure 2) about tR = 40 min (Figure 5). The maximum peak capacity of up to 1200 bar. isocratic conditions (Figure 1 and Figure 2). the Hypersil GOLD column was probably not achieved but

Results: Exploiting the maximum column pressure, the FIGURE 1: Efficiencies of columns with different found to be approximately nc = 150 at a retention time of p g p ,best peak-capacity-to-analysis-time ratio for isocratic stationary phase architectures but the same

y cabout tR = 25 min (Figure 5). The flow-pressure range was p p y y

conditions was determined by Thermo Scientific™y p

dimension of 2.1 × 100 mm.R ( g ) p g

similar for both column-series (Figure 6).conditions was determined by Thermo ScientificAccucore™ columns 2 6 μm particle size With gradient

dimension of 2.1 100 mm.Poro s Particles d 2 5 m C18 Comp Wm

)

similar for both column series (Figure 6).Accucore columns, 2.6 μm particle size. With gradient conditions Thermo Scientific™ Hypersil GOLD™ columns

Porous Particles, dp= 2.5 μm, C18, Comp. WPorous Particles d = 1 9 μm C18 Hypersil GOLD19(µ

m Traditional Chinese Medicine (TCM)conditions, Thermo Scientific Hypersil GOLD columns, 1 9 μm particle size were found to be superior

Porous Particles, dp= 1.9 μm, C18, Hypersil GOLDSolid Core Particles, dp= 2.6 μm, C18, Accucore17H

(

The series of Hypersil GOLD (2 1 × 300 mm) columns and1.9 μm particle size, were found to be superior. Solid Core Particles, dp 2.6 μm, C18, AccucoreSolid Core Particles, dp= 2.7 μm, C18, Comp. A15

17

ght The series of Hypersil GOLD (2.1 300 mm) columns and

Accucore (2 1 × 450 mm) columns were tested by using the

I t d tip μ p

Monolithic Rod, C18(e), Comp. M1315

eig

ne

Accucore (2.1 × 450 mm) columns were tested by using the TCM sample “Dang Gui Long Hui Wan“ under gradientIntroduction 13

e H

eyl

en TCM-sample Dang Gui Long Hui Wan under gradient conditions Applying the optimum linear velocity to each the

Th ti f ll l d ith i t11la

teo-

Xy conditions. Applying the optimum linear velocity to each, the th A l i i t d k itThe separation of all sample compounds with appropriate 9lP

lof

o three Accucore columns in series generated a peak capacity resolution in the shortest time possible is a common goal 7ic

al o of nc = 670 at the gradient time tg = 60 min with a of most chromatographers. This goal becomes even more 5re

ti gbackpressure of 850 bar. The two Hypersil GOLD columns in g g

relevant with highly complex samples. Generally, highest 35

eor

series generated a peak capacity of nc = 480 at the gradient g y p p y, gpeak capacities are achieved by employing an additional

30.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5Th

e g p p y c gtime tg = 28 min with a backpressure of 1200 bar.peak capacities are achieved by employing an additional

chromatographic dimension Dedicated instrumentation Linear Velocity u (mm/s)time tg 28 min with a backpressure of 1200 bar.

chromatographic dimension. Dedicated instrumentation, extensive method development and a proper combination FIGURE 2: Backpressures of columns with different FIGURE 7: Calculated peak capacities with columnextensive method development, and a proper combination of separation chemistries are necessary in this case The

FIGURE 2: Backpressures of columns with different t ti h hit t b t th

FIGURE 7: Calculated peak capacities with column chains in gradient mode: Hypersil GOLD 2 1 × 300 mmof separation chemistries are necessary in this case. The

theoretical basis has been summarized by Guiochon 1stationary phase architectures but the same chains in gradient mode: Hypersil GOLD 2.1 × 300 mm

l A 2 1 × 450 ltheoretical basis has been summarized by Guiochon.1 dimension of 2.1 × 100 mm. column or Accucore 2.1 × 450 mm column.

Highest resolution in shortest time can also be achieved Porous Particles d = 2 5 μm1000 700Highest resolution in shortest time can also be achieved by maximizing the peak-capacity-to-analysis-time ratio of

Porous Particles, dp 2.5 μmPorous Particles, dp= 1.9 μm F 0 45 0 65 0 60 0 45600

700

yby maximizing the peak-capacity-to-analysis-time ratio of an application This approach is easily implemented by

, p μSolid Core Particles, dp= 2.7 μm800) (mL/min)

0.45 0.65 0.60 0.45500600

city

an application. This approach is easily implemented by applying columns of extended length packed with porous

pSolid Core Particles, dp= 2.6 μmM lithi R dba

r)

tG 40 42 28 60500pa

c

applying columns of extended length packed with porous b 2 i ti l O th th h d h t

Monolithic Rod600e(b tG

(min)40 42 28 60400C

a

sub-2-micron particles. On the other hand, such a setup ure ( )

Pak

lead to a column head pressure up to 1200 bar and even 400ess P

(bar)930 1200 1200 850200P

e

more. In this work, experimental results from the use of Max Pressure of MonolithPr

e (bar)

n 430 600 480 670different stationary phase architectures in column lengths Max. Pressure of Monolith200

P nc 430 600 480 6700y p g

of 100 mm to 450 mm and at pressures up to 1200 bar are0

of 100 mm to 450 mm and at pressures up to 1200 bar are discussed Furthermore it is evaluated which approach 0 FIGURE 8: Ratio of peak-capacity-to-analysis-time with discussed. Furthermore, it is evaluated which approach provides the best peak-capacity-to-analysis-time ratio for

0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05Fl t F ( L/ i )

p p y ycolumn chains in gradient mode: Hypersil GOLD 2.1 ×provides the best peak-capacity-to-analysis-time ratio for

the analysis of complex sample mixturesFlow rate F (mL/min) g yp

300 mm column or Accucore 2.1 × 450 mm column.the analysis of complex sample mixtures. In one dimensional liquid chromatography the best peak

300 mm column or Accucore 2.1 450 mm column.17

M th dIn one-dimensional liquid chromatography, the best peak-

it t l i ti ti i hi d b l iti17

o FMethods capacity-to-analysis-time ratio is achieved by exploiting 14to

Rat

io F (mL/min)

0.45 0.65 0.60 0.45

Liquid Chromatography System the flow-pressure footprint of the system and column 11ci

ty

e R (mL/min)

/tLiquid Chromatography System™ ™ ™

hardware. High peak capacity is basically achieved 11

pac

Tim

e nc/tG( / )

11 14 17 11Thermo Scientific ™ Dionex™ UltiMate™ 3000 XRS system: through a high number of theoretical plates (N) per C

ap s T (1/min)

solvent rack SR-3000, pump LPG-3400XRS, sampler g g p ( ) p

column. The optimum linear velocity is determined by the 5ak C

lysi P 930 1200 1200 850p p p

WPS-3000TXRS, column thermostat TCC-3000RS, column. The optimum linear velocity is determined by the minimum theoretical plate height (Figure 1) Extending the P

eaA

nal

(bar)930 1200 1200 850

, ,and detector DAD-3000RS.

minimum theoretical plate height (Figure 1). Extending the column length means enhancing the column efficiency (N) 0

Aand detector DAD 3000RS. column length means enhancing the column efficiency (N) but also requires a higher pressure to achieve the same

0

Data Analysis but also requires a higher pressure to achieve the same linear velocity (Figure 2) The Accucore column chain provided higher peak capacities

Thermo Scientific™ Dionex™ Chromeleon™linear velocity (Figure 2). The Accucore column chain provided higher peak capacities

th th H il GOLD l h i t h fl tThermo Scientific Dionex ChromeleonChromatograph Data S stem Soft are ersion 6 8 Different concepts of stationary phase design were than the Hypersil GOLD column chain at each flow rate Chromatography Data System Software version 6.8 p y p g

compared by using identical column dimensions. The (Figure 7). The ratio between the peak capacities of both Comparison of 2 1x100 mm columns

p y gmonolithic rod showed the poorest efficiency but also column chains was the same as found under isocratic Comparison of 2.1x100 mm columns

C l Diff t d i f t ti h ( llmonolithic rod showed the poorest efficiency but also required the lowest flow-pressure footprint The maximum conditions by using phenones (Figure 5). But when

Columns: Different designs of stationary phase (all required the lowest flow-pressure footprint. The maximum operation pressure of a monolithic rod is about 200 bar

y g p ( g )comparing the ratio of peak capacity to analysis time, the

RP-C18) with 2.1 × 100 mm each operation pressure of a monolithic rod is about 200 bar and was almost reached for the 2 1 x 100 mm column at a

comparing the ratio of peak capacity to analysis time, the Accucore column chain generated 14 peaks per minute while

RP-5 Standard: Theophylline, p-nitroaniline, phenetole, and was almost reached for the 2.1 x 100 mm column at a fl t f 1 L/ i (Fi 2) H li

Accucore column chain generated 14 peaks per minute while the Hypersil GOLD column chain generated 17 peak per

o-xylene, and methyl benzoate flow rate of 1 mL/min (Figure 2). Hence, coupling li hi d h i hi h ld h l

the Hypersil GOLD column chain generated 17 peak per minute at 1200 bar backpressure (Figure 8) Thus byy , y

Mobile Phase A: Fisher Scientific™ Optima™ LC/MS water monolithic rods to a chain which would show an equal minute at 1200 bar backpressure (Figure 8). Thus, by exhausting the maximum pressure the Accucore chain wasMobile Phase A: Fisher Scientific Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrile number of theoretical plates compared to the other column exhausting the maximum pressure, the Accucore chain was bl t hi th hi h t k it b t th H ilMobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% B types is not feasible. able to achieve the highest peak capacity but the Hypersil OIsocratic Conditions: 50% A and 50% B

Flow Rate: 0 1 1 0 mL/minypHypersil GOLD and Accucore columns were found to

GOLD column chain provided the maximum ratio of peak Flow Rate: 0.1–1.0 mL/minInjection Volume:1 µL

Hypersil GOLD and Accucore columns were found to provide the highest column efficiency (Figure 1)

capacity to analysis time.Injection Volume:1 µLC l T t 30 °C

provide the highest column efficiency (Figure 1). F th th A l h d b t 60% fColumn Temperature: 30 °C Furthermore, the Accucore columns showed about 60% of th b k l ti t th H il GOLD l ConclusionDetection wavelength: 254 nm the backpressure relative to the Hypersil GOLD columns Conclusion

Detection Rate: 20 Hz at identical flow rates (Figure 2). Accordingly, to exploit the By exploiting the maximum pressure of the column Comparison of columns coupled in series flow-pressure footprint of the system and column

y p g phardware, a 2.1 × 450 mm long chain of Accucore C18Comparison of columns coupled in series

C l S i H il GOLD C18

p p yhardware, three Accucore 2.1 × 150 mm columns were

hardware, a 2.1 450 mm long chain of Accucore C18 (2 6 μm) columns could be built It produced a higherColumn-Series: Hypersil GOLD C18, ,

coupled in series to a sequence of 2.1 × 450 mm. For(2.6 μm) columns could be built. It produced a higher efficiency (N) than the 2 1 × 300 mm long Hypersil GOLD1.9 μm, 2.1 × 200 and 2.1 × 100 mm coupled in series to a sequence of 2.1 450 mm. For

Hypersil GOLD columns 2 1 × 200 mm and 2 1 × 100efficiency (N) than the 2.1 × 300 mm long Hypersil GOLD (1 9 μm) column chainAccucore C18, Hypersil GOLD columns , 2.1 × 200 mm and 2.1 × 100

mm were coupled to a sequence of 2 1 × 300 mm(1.9 μm) column chain.

2.6 μm, 2.1 × 150 mm (three) mm were coupled to a sequence of 2.1 × 300 mm. On the Accucore 2.1 × 450 mm column chain, a peakμ , ( )

Sample: Phenones and uracil Comparison of Columns Coupled in SeriesOn the Accucore 2.1 450 mm column chain, a peak capacity of 600 was achieved for the TCM “Dang GuiSample: Phenones and uracil

Mobile Phase A: Optima LC/MS waterp p

The series of Hypersil GOLD (2 1 × 300 mm) andcapacity of 600 was achieved for the TCM Dang Gui Long Hui Wan” with 42 min gradient time (14 peaks perMobile Phase A: Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrileThe series of Hypersil GOLD (2.1 × 300 mm) and Acc core (2 1 × 450 mm) col mns ere characteri ed

Long Hui Wan with 42 min gradient time (14 peaks per minute) A peak capacity of 670 could be realized with 60Mobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% BAccucore (2.1 × 450 mm) columns were characterized

ith lk l h t d d d i ti ditiminute). A peak capacity of 670 could be realized with 60

i di t ti hi h i d th k itIsocratic Conditions: 50% A and 50% BFlow Rate: 0 05 0 5 mL/min

with alkylphenone standard under isocratic conditions. min gradient time, which compromised the peak-capacity-t l i ti ti (11 k i t )Flow Rate: 0.05–0.5 mL/min

I j ti V l 5 L FIGURE 4: TheoreticalFIGURE 3 Theoretical to-analysis-time ratio (11 peaks per minute).Injection Volume: 5 μL FIGURE 4: Theoretical

plate numbers (N) ofFIGURE 3. Theoretical plate heights (H) of On the Hypersil GOLD 2 1 × 300 mm column chain aColumn Temperature: 35 °C plate numbers (N) of

hexanophenoneplate heights (H) of hexanophenone

On the Hypersil GOLD 2.1 × 300 mm column chain, a peak capacity of 430 was achieved for the TCM “DangDetection: 250 nm hexanophenone.hexanophenone. peak capacity of 430 was achieved for the TCM Dang G i L H i W “ i 40 i di t ti (11 kDetection Rate: 20 Hz N 100 x10³20m

)

Hypersil GOLD 2 1 x 300 mmGui Long Hui Wan“ in 40 min gradient time (11 peaks per

i t ) A k it f 480 d t i d ith 28T diti l Chi M di i (TCM) es 80

20

(µm

A 2 1 450

Hypersil GOLD 2.1 x 300 mm minute). A peak capacity of 480 was determined with 28 Traditional Chinese Medicine (TCM)

plat 80

16H ( Accucore 2.1 x 450 mm min gradient time, which benefited the peak-capacity-to-

Columns: see above

ofp

6016

ght analysis-time ratio (17 peaks per minute).

TCM-Sample: Dang Gui Long Hui Wan Nr. 3560 B, ero

4010Hei

g y ( p p )

M lithi l t id hi h k itiHerba Natura Harald Noll BV, Dinxperlo, the Netherlands mbe 4010

e H Monolithic columns cannot provide high peak capacities

i bl d h t ti d t th i i, p ,

Sample Preparation: 200 mg of sample mixed with 10 Num 204la

te in a comparable and short time due to their maximum Sample Preparation: 200 mg of sample mixed with 10 mL water sonicated for 15 min and filtered through

N 20

0 1 0 3 0 540 5 2 5 4 5

P operating pressure of 200 bar.mL water, sonicated for 15 min, and filtered through 0 2 μm PVDF membrane Flow rate F (mL/min)

0.1 0.3 0.50.5 2.5 4.5Linear Velocity u (mm/s)0.2 μm PVDF membrane

Mobile Phase A: 50 mM NH OAc pH 4 8Flow rate F (mL/min)Linear Velocity u (mm/s) ReferencesMobile Phase A: 50 mM NH4OAc, pH 4.8

Mobile Phase B: Optima LC/MS acetonitrile FIGURE 5 Peak capacities FIGURE 6: SystemReferences

1 G i h G Th Li it f th S ti P fMobile Phase B: Optima LC/MS acetonitrileG di t F 5% B t 50% B

FIGURE 5. Peak capacities (valid for all alkylphenones)

FIGURE 6: System backpressures

1. Guiochon, G. The Limits of the Separation Power of Gradient : From 5% B to 50% B, (valid for all alkylphenones). backpressures. Unidimensional Column liquid chromatography, Gradient Time: see figure (mentioned as tG)

1200J. Chromatogr. A, 2006, 1126, 6-49.

Flow Rate: see figure 220n c 1200) © 2014 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific IncInjection Volume: 2 μL ity ba

r © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe j μ

Column Temperature: 45 °C 180paci 800e(b the intellectual property rights of others.

PO71312-EN 0814SColumn Temperature: 45 CDetection wavelength: 280 nm C

ap sure

Detection wavelength: 280 nmDetection Rate: 50 Hz 140k

C

400ess

Detection Rate: 50 Hz 140

Pea 400

Pre

100

P

020 40 60 80R t ti Ti t ( i )

0 0.3 0.50.1Retention Time tR (min) Flow rate F (mL/min)

6 Maximizing the Peak-Capacity-to-Analysis-Time Ratio in One-Dimensional Liquid Chromatography

Maximizing the Peak Capacity to Analysis Time Ratio in One DimensionalMaximizing the Peak-Capacity-to-Analysis-Time Ratio in One-Dimensional a g t e ea Capac ty to a ys s e at o O e e s o aCLiquid ChromatographyLiquid Chromatography

Mi h l H id S F b l d F k St iMichael Heidorn, Susanne Fabel and Frank SteinerMichael Heidorn, Susanne Fabel and Frank SteinerTh Fi h S i tifi G i GThermo Fisher Scientific, Germering, GermanyThermo Fisher Scientific, Germering, Germany

Overview Results While the theoretical plate heights (Figure 3) of both column-Overview Results p g ( g )series merged each other the same way as it was already

Purpose: A separation of complex sample mixtures with Comparison of 2 1 × 100 mm Columnsseries merged each other the same way as it was already seen in figure 1 the number of theoretical plates (Figure 4)Purpose: A separation of complex sample mixtures with

appropriate resolution in the shortest time possibleComparison of 2.1 × 100 mm Columns seen in figure 1, the number of theoretical plates (Figure 4)

was much higher for the longer sequence of Accucoreappropriate resolution in the shortest time possible. Different chromatographic columns (porous C18-silica, was much higher for the longer sequence of Accucore columns The maximum peak capacity of the AccucoreMethods: Different stationary phase architectures, column

g (fused-core silica-based C18, and monolithic silica-based columns. The maximum peak capacity of the Accucore

column chain was found to be n 220 at a retention time ofy p ,lengths, and temperatures, as well as applying pressures

,C18e) were tested by using the RP-5 standard under column chain was found to be nc = 220 at a retention time of

b t t 40 i (Fi 5) Th i k it flengths, and temperatures, as well as applying pressures up to 1200 bar

C18e) were tested by using the RP 5 standard under isocratic conditions (Figure 1 and Figure 2) about tR = 40 min (Figure 5). The maximum peak capacity of up to 1200 bar. isocratic conditions (Figure 1 and Figure 2). the Hypersil GOLD column was probably not achieved but

Results: Exploiting the maximum column pressure, the FIGURE 1: Efficiencies of columns with different found to be approximately nc = 150 at a retention time of p g p ,best peak-capacity-to-analysis-time ratio for isocratic stationary phase architectures but the same

y cabout tR = 25 min (Figure 5). The flow-pressure range was p p y y

conditions was determined by Thermo Scientific™y p

dimension of 2.1 × 100 mm.R ( g ) p g

similar for both column-series (Figure 6).conditions was determined by Thermo ScientificAccucore™ columns 2 6 μm particle size With gradient

dimension of 2.1 100 mm.Poro s Particles d 2 5 m C18 Comp Wm

)

similar for both column series (Figure 6).Accucore columns, 2.6 μm particle size. With gradient conditions Thermo Scientific™ Hypersil GOLD™ columns

Porous Particles, dp= 2.5 μm, C18, Comp. WPorous Particles d = 1 9 μm C18 Hypersil GOLD19(µ

m Traditional Chinese Medicine (TCM)conditions, Thermo Scientific Hypersil GOLD columns, 1 9 μm particle size were found to be superior

Porous Particles, dp= 1.9 μm, C18, Hypersil GOLDSolid Core Particles, dp= 2.6 μm, C18, Accucore17H

(

The series of Hypersil GOLD (2 1 × 300 mm) columns and1.9 μm particle size, were found to be superior. Solid Core Particles, dp 2.6 μm, C18, AccucoreSolid Core Particles, dp= 2.7 μm, C18, Comp. A15

17

ght The series of Hypersil GOLD (2.1 300 mm) columns and

Accucore (2 1 × 450 mm) columns were tested by using the

I t d tip μ p

Monolithic Rod, C18(e), Comp. M1315

eig

ne

Accucore (2.1 × 450 mm) columns were tested by using the TCM sample “Dang Gui Long Hui Wan“ under gradientIntroduction 13

e H

eyl

en TCM-sample Dang Gui Long Hui Wan under gradient conditions Applying the optimum linear velocity to each the

Th ti f ll l d ith i t11la

teo-

Xy conditions. Applying the optimum linear velocity to each, the th A l i i t d k itThe separation of all sample compounds with appropriate 9lP

lof

o three Accucore columns in series generated a peak capacity resolution in the shortest time possible is a common goal 7ic

al o of nc = 670 at the gradient time tg = 60 min with a of most chromatographers. This goal becomes even more 5re

ti gbackpressure of 850 bar. The two Hypersil GOLD columns in g g

relevant with highly complex samples. Generally, highest 35

eor

series generated a peak capacity of nc = 480 at the gradient g y p p y, gpeak capacities are achieved by employing an additional

30.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5Th

e g p p y c gtime tg = 28 min with a backpressure of 1200 bar.peak capacities are achieved by employing an additional

chromatographic dimension Dedicated instrumentation Linear Velocity u (mm/s)time tg 28 min with a backpressure of 1200 bar.

chromatographic dimension. Dedicated instrumentation, extensive method development and a proper combination FIGURE 2: Backpressures of columns with different FIGURE 7: Calculated peak capacities with columnextensive method development, and a proper combination of separation chemistries are necessary in this case The

FIGURE 2: Backpressures of columns with different t ti h hit t b t th

FIGURE 7: Calculated peak capacities with column chains in gradient mode: Hypersil GOLD 2 1 × 300 mmof separation chemistries are necessary in this case. The

theoretical basis has been summarized by Guiochon 1stationary phase architectures but the same chains in gradient mode: Hypersil GOLD 2.1 × 300 mm

l A 2 1 × 450 ltheoretical basis has been summarized by Guiochon.1 dimension of 2.1 × 100 mm. column or Accucore 2.1 × 450 mm column.

Highest resolution in shortest time can also be achieved Porous Particles d = 2 5 μm1000 700Highest resolution in shortest time can also be achieved by maximizing the peak-capacity-to-analysis-time ratio of

Porous Particles, dp 2.5 μmPorous Particles, dp= 1.9 μm F 0 45 0 65 0 60 0 45600

700

yby maximizing the peak-capacity-to-analysis-time ratio of an application This approach is easily implemented by

, p μSolid Core Particles, dp= 2.7 μm800) (mL/min)

0.45 0.65 0.60 0.45500600

city

an application. This approach is easily implemented by applying columns of extended length packed with porous

pSolid Core Particles, dp= 2.6 μmM lithi R dba

r)

tG 40 42 28 60500pa

c

applying columns of extended length packed with porous b 2 i ti l O th th h d h t

Monolithic Rod600e(b tG

(min)40 42 28 60400C

a

sub-2-micron particles. On the other hand, such a setup ure ( )

Pak

lead to a column head pressure up to 1200 bar and even 400ess P

(bar)930 1200 1200 850200P

e

more. In this work, experimental results from the use of Max Pressure of MonolithPr

e (bar)

n 430 600 480 670different stationary phase architectures in column lengths Max. Pressure of Monolith200

P nc 430 600 480 6700y p g

of 100 mm to 450 mm and at pressures up to 1200 bar are0

of 100 mm to 450 mm and at pressures up to 1200 bar are discussed Furthermore it is evaluated which approach 0 FIGURE 8: Ratio of peak-capacity-to-analysis-time with discussed. Furthermore, it is evaluated which approach provides the best peak-capacity-to-analysis-time ratio for

0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05Fl t F ( L/ i )

p p y ycolumn chains in gradient mode: Hypersil GOLD 2.1 ×provides the best peak-capacity-to-analysis-time ratio for

the analysis of complex sample mixturesFlow rate F (mL/min) g yp

300 mm column or Accucore 2.1 × 450 mm column.the analysis of complex sample mixtures. In one dimensional liquid chromatography the best peak

300 mm column or Accucore 2.1 450 mm column.17

M th dIn one-dimensional liquid chromatography, the best peak-

it t l i ti ti i hi d b l iti17

o FMethods capacity-to-analysis-time ratio is achieved by exploiting 14to

Rat

io F (mL/min)

0.45 0.65 0.60 0.45

Liquid Chromatography System the flow-pressure footprint of the system and column 11ci

ty

e R (mL/min)

/tLiquid Chromatography System™ ™ ™

hardware. High peak capacity is basically achieved 11

pac

Tim

e nc/tG( / )

11 14 17 11Thermo Scientific ™ Dionex™ UltiMate™ 3000 XRS system: through a high number of theoretical plates (N) per C

ap s T (1/min)

solvent rack SR-3000, pump LPG-3400XRS, sampler g g p ( ) p

column. The optimum linear velocity is determined by the 5ak C

lysi P 930 1200 1200 850p p p

WPS-3000TXRS, column thermostat TCC-3000RS, column. The optimum linear velocity is determined by the minimum theoretical plate height (Figure 1) Extending the P

eaA

nal

(bar)930 1200 1200 850

, ,and detector DAD-3000RS.

minimum theoretical plate height (Figure 1). Extending the column length means enhancing the column efficiency (N) 0

Aand detector DAD 3000RS. column length means enhancing the column efficiency (N) but also requires a higher pressure to achieve the same

0

Data Analysis but also requires a higher pressure to achieve the same linear velocity (Figure 2) The Accucore column chain provided higher peak capacities

Thermo Scientific™ Dionex™ Chromeleon™linear velocity (Figure 2). The Accucore column chain provided higher peak capacities

th th H il GOLD l h i t h fl tThermo Scientific Dionex ChromeleonChromatograph Data S stem Soft are ersion 6 8 Different concepts of stationary phase design were than the Hypersil GOLD column chain at each flow rate Chromatography Data System Software version 6.8 p y p g

compared by using identical column dimensions. The (Figure 7). The ratio between the peak capacities of both Comparison of 2 1x100 mm columns

p y gmonolithic rod showed the poorest efficiency but also column chains was the same as found under isocratic Comparison of 2.1x100 mm columns

C l Diff t d i f t ti h ( llmonolithic rod showed the poorest efficiency but also required the lowest flow-pressure footprint The maximum conditions by using phenones (Figure 5). But when

Columns: Different designs of stationary phase (all required the lowest flow-pressure footprint. The maximum operation pressure of a monolithic rod is about 200 bar

y g p ( g )comparing the ratio of peak capacity to analysis time, the

RP-C18) with 2.1 × 100 mm each operation pressure of a monolithic rod is about 200 bar and was almost reached for the 2 1 x 100 mm column at a

comparing the ratio of peak capacity to analysis time, the Accucore column chain generated 14 peaks per minute while

RP-5 Standard: Theophylline, p-nitroaniline, phenetole, and was almost reached for the 2.1 x 100 mm column at a fl t f 1 L/ i (Fi 2) H li

Accucore column chain generated 14 peaks per minute while the Hypersil GOLD column chain generated 17 peak per

o-xylene, and methyl benzoate flow rate of 1 mL/min (Figure 2). Hence, coupling li hi d h i hi h ld h l

the Hypersil GOLD column chain generated 17 peak per minute at 1200 bar backpressure (Figure 8) Thus byy , y

Mobile Phase A: Fisher Scientific™ Optima™ LC/MS water monolithic rods to a chain which would show an equal minute at 1200 bar backpressure (Figure 8). Thus, by exhausting the maximum pressure the Accucore chain wasMobile Phase A: Fisher Scientific Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrile number of theoretical plates compared to the other column exhausting the maximum pressure, the Accucore chain was bl t hi th hi h t k it b t th H ilMobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% B types is not feasible. able to achieve the highest peak capacity but the Hypersil OIsocratic Conditions: 50% A and 50% B

Flow Rate: 0 1 1 0 mL/minypHypersil GOLD and Accucore columns were found to

GOLD column chain provided the maximum ratio of peak Flow Rate: 0.1–1.0 mL/minInjection Volume:1 µL

Hypersil GOLD and Accucore columns were found to provide the highest column efficiency (Figure 1)

capacity to analysis time.Injection Volume:1 µLC l T t 30 °C

provide the highest column efficiency (Figure 1). F th th A l h d b t 60% fColumn Temperature: 30 °C Furthermore, the Accucore columns showed about 60% of th b k l ti t th H il GOLD l ConclusionDetection wavelength: 254 nm the backpressure relative to the Hypersil GOLD columns Conclusion

Detection Rate: 20 Hz at identical flow rates (Figure 2). Accordingly, to exploit the By exploiting the maximum pressure of the column Comparison of columns coupled in series flow-pressure footprint of the system and column

y p g phardware, a 2.1 × 450 mm long chain of Accucore C18Comparison of columns coupled in series

C l S i H il GOLD C18

p p yhardware, three Accucore 2.1 × 150 mm columns were

hardware, a 2.1 450 mm long chain of Accucore C18 (2 6 μm) columns could be built It produced a higherColumn-Series: Hypersil GOLD C18, ,

coupled in series to a sequence of 2.1 × 450 mm. For(2.6 μm) columns could be built. It produced a higher efficiency (N) than the 2 1 × 300 mm long Hypersil GOLD1.9 μm, 2.1 × 200 and 2.1 × 100 mm coupled in series to a sequence of 2.1 450 mm. For

Hypersil GOLD columns 2 1 × 200 mm and 2 1 × 100efficiency (N) than the 2.1 × 300 mm long Hypersil GOLD (1 9 μm) column chainAccucore C18, Hypersil GOLD columns , 2.1 × 200 mm and 2.1 × 100

mm were coupled to a sequence of 2 1 × 300 mm(1.9 μm) column chain.

2.6 μm, 2.1 × 150 mm (three) mm were coupled to a sequence of 2.1 × 300 mm. On the Accucore 2.1 × 450 mm column chain, a peakμ , ( )

Sample: Phenones and uracil Comparison of Columns Coupled in SeriesOn the Accucore 2.1 450 mm column chain, a peak capacity of 600 was achieved for the TCM “Dang GuiSample: Phenones and uracil

Mobile Phase A: Optima LC/MS waterp p

The series of Hypersil GOLD (2 1 × 300 mm) andcapacity of 600 was achieved for the TCM Dang Gui Long Hui Wan” with 42 min gradient time (14 peaks perMobile Phase A: Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrileThe series of Hypersil GOLD (2.1 × 300 mm) and Acc core (2 1 × 450 mm) col mns ere characteri ed

Long Hui Wan with 42 min gradient time (14 peaks per minute) A peak capacity of 670 could be realized with 60Mobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% BAccucore (2.1 × 450 mm) columns were characterized

ith lk l h t d d d i ti ditiminute). A peak capacity of 670 could be realized with 60

i di t ti hi h i d th k itIsocratic Conditions: 50% A and 50% BFlow Rate: 0 05 0 5 mL/min

with alkylphenone standard under isocratic conditions. min gradient time, which compromised the peak-capacity-t l i ti ti (11 k i t )Flow Rate: 0.05–0.5 mL/min

I j ti V l 5 L FIGURE 4: TheoreticalFIGURE 3 Theoretical to-analysis-time ratio (11 peaks per minute).Injection Volume: 5 μL FIGURE 4: Theoretical

plate numbers (N) ofFIGURE 3. Theoretical plate heights (H) of On the Hypersil GOLD 2 1 × 300 mm column chain aColumn Temperature: 35 °C plate numbers (N) of

hexanophenoneplate heights (H) of hexanophenone

On the Hypersil GOLD 2.1 × 300 mm column chain, a peak capacity of 430 was achieved for the TCM “DangDetection: 250 nm hexanophenone.hexanophenone. peak capacity of 430 was achieved for the TCM Dang G i L H i W “ i 40 i di t ti (11 kDetection Rate: 20 Hz N 100 x10³20m

)

Hypersil GOLD 2 1 x 300 mmGui Long Hui Wan“ in 40 min gradient time (11 peaks per

i t ) A k it f 480 d t i d ith 28T diti l Chi M di i (TCM) es 80

20

(µm

A 2 1 450

Hypersil GOLD 2.1 x 300 mm minute). A peak capacity of 480 was determined with 28 Traditional Chinese Medicine (TCM)

plat 80

16H ( Accucore 2.1 x 450 mm min gradient time, which benefited the peak-capacity-to-

Columns: see above

ofp

6016

ght analysis-time ratio (17 peaks per minute).

TCM-Sample: Dang Gui Long Hui Wan Nr. 3560 B, ero

4010Hei

g y ( p p )

M lithi l t id hi h k itiHerba Natura Harald Noll BV, Dinxperlo, the Netherlands mbe 4010

e H Monolithic columns cannot provide high peak capacities

i bl d h t ti d t th i i, p ,

Sample Preparation: 200 mg of sample mixed with 10 Num 204la

te in a comparable and short time due to their maximum Sample Preparation: 200 mg of sample mixed with 10 mL water sonicated for 15 min and filtered through

N 20

0 1 0 3 0 540 5 2 5 4 5

P operating pressure of 200 bar.mL water, sonicated for 15 min, and filtered through 0 2 μm PVDF membrane Flow rate F (mL/min)

0.1 0.3 0.50.5 2.5 4.5Linear Velocity u (mm/s)0.2 μm PVDF membrane

Mobile Phase A: 50 mM NH OAc pH 4 8Flow rate F (mL/min)Linear Velocity u (mm/s) ReferencesMobile Phase A: 50 mM NH4OAc, pH 4.8

Mobile Phase B: Optima LC/MS acetonitrile FIGURE 5 Peak capacities FIGURE 6: SystemReferences

1 G i h G Th Li it f th S ti P fMobile Phase B: Optima LC/MS acetonitrileG di t F 5% B t 50% B

FIGURE 5. Peak capacities (valid for all alkylphenones)

FIGURE 6: System backpressures

1. Guiochon, G. The Limits of the Separation Power of Gradient : From 5% B to 50% B, (valid for all alkylphenones). backpressures. Unidimensional Column liquid chromatography, Gradient Time: see figure (mentioned as tG)

1200J. Chromatogr. A, 2006, 1126, 6-49.

Flow Rate: see figure 220n c 1200) © 2014 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific IncInjection Volume: 2 μL ity ba

r © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe j μ

Column Temperature: 45 °C 180paci 800e(b the intellectual property rights of others.

PO71312-EN 0814SColumn Temperature: 45 CDetection wavelength: 280 nm C

ap sure

Detection wavelength: 280 nmDetection Rate: 50 Hz 140k

C

400ess

Detection Rate: 50 Hz 140

Pea 400

Pre

100

P

020 40 60 80R t ti Ti t ( i )

0 0.3 0.50.1Retention Time tR (min) Flow rate F (mL/min)

Maximizing the Peak Capacity to Analysis Time Ratio in One DimensionalMaximizing the Peak-Capacity-to-Analysis-Time Ratio in One-Dimensional a g t e ea Capac ty to a ys s e at o O e e s o aCLiquid ChromatographyLiquid Chromatography

Mi h l H id S F b l d F k St iMichael Heidorn, Susanne Fabel and Frank SteinerMichael Heidorn, Susanne Fabel and Frank SteinerTh Fi h S i tifi G i GThermo Fisher Scientific, Germering, GermanyThermo Fisher Scientific, Germering, Germany

Overview Results While the theoretical plate heights (Figure 3) of both column-Overview Results p g ( g )series merged each other the same way as it was already

Purpose: A separation of complex sample mixtures with Comparison of 2 1 × 100 mm Columnsseries merged each other the same way as it was already seen in figure 1 the number of theoretical plates (Figure 4)Purpose: A separation of complex sample mixtures with

appropriate resolution in the shortest time possibleComparison of 2.1 × 100 mm Columns seen in figure 1, the number of theoretical plates (Figure 4)

was much higher for the longer sequence of Accucoreappropriate resolution in the shortest time possible. Different chromatographic columns (porous C18-silica, was much higher for the longer sequence of Accucore columns The maximum peak capacity of the AccucoreMethods: Different stationary phase architectures, column

g (fused-core silica-based C18, and monolithic silica-based columns. The maximum peak capacity of the Accucore

column chain was found to be n 220 at a retention time ofy p ,lengths, and temperatures, as well as applying pressures

,C18e) were tested by using the RP-5 standard under column chain was found to be nc = 220 at a retention time of

b t t 40 i (Fi 5) Th i k it flengths, and temperatures, as well as applying pressures up to 1200 bar

C18e) were tested by using the RP 5 standard under isocratic conditions (Figure 1 and Figure 2) about tR = 40 min (Figure 5). The maximum peak capacity of up to 1200 bar. isocratic conditions (Figure 1 and Figure 2). the Hypersil GOLD column was probably not achieved but

Results: Exploiting the maximum column pressure, the FIGURE 1: Efficiencies of columns with different found to be approximately nc = 150 at a retention time of p g p ,best peak-capacity-to-analysis-time ratio for isocratic stationary phase architectures but the same

y cabout tR = 25 min (Figure 5). The flow-pressure range was p p y y

conditions was determined by Thermo Scientific™y p

dimension of 2.1 × 100 mm.R ( g ) p g

similar for both column-series (Figure 6).conditions was determined by Thermo ScientificAccucore™ columns 2 6 μm particle size With gradient

dimension of 2.1 100 mm.Poro s Particles d 2 5 m C18 Comp Wm

)

similar for both column series (Figure 6).Accucore columns, 2.6 μm particle size. With gradient conditions Thermo Scientific™ Hypersil GOLD™ columns

Porous Particles, dp= 2.5 μm, C18, Comp. WPorous Particles d = 1 9 μm C18 Hypersil GOLD19(µ

m Traditional Chinese Medicine (TCM)conditions, Thermo Scientific Hypersil GOLD columns, 1 9 μm particle size were found to be superior

Porous Particles, dp= 1.9 μm, C18, Hypersil GOLDSolid Core Particles, dp= 2.6 μm, C18, Accucore17H

(

The series of Hypersil GOLD (2 1 × 300 mm) columns and1.9 μm particle size, were found to be superior. Solid Core Particles, dp 2.6 μm, C18, AccucoreSolid Core Particles, dp= 2.7 μm, C18, Comp. A15

17

ght The series of Hypersil GOLD (2.1 300 mm) columns and

Accucore (2 1 × 450 mm) columns were tested by using the

I t d tip μ p

Monolithic Rod, C18(e), Comp. M1315

eig

ne

Accucore (2.1 × 450 mm) columns were tested by using the TCM sample “Dang Gui Long Hui Wan“ under gradientIntroduction 13

e H

eyl

en TCM-sample Dang Gui Long Hui Wan under gradient conditions Applying the optimum linear velocity to each the

Th ti f ll l d ith i t11la

teo-

Xy conditions. Applying the optimum linear velocity to each, the th A l i i t d k itThe separation of all sample compounds with appropriate 9lP

lof

o three Accucore columns in series generated a peak capacity resolution in the shortest time possible is a common goal 7ic

al o of nc = 670 at the gradient time tg = 60 min with a of most chromatographers. This goal becomes even more 5re

ti gbackpressure of 850 bar. The two Hypersil GOLD columns in g g

relevant with highly complex samples. Generally, highest 35

eor

series generated a peak capacity of nc = 480 at the gradient g y p p y, gpeak capacities are achieved by employing an additional

30.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5Th

e g p p y c gtime tg = 28 min with a backpressure of 1200 bar.peak capacities are achieved by employing an additional

chromatographic dimension Dedicated instrumentation Linear Velocity u (mm/s)time tg 28 min with a backpressure of 1200 bar.

chromatographic dimension. Dedicated instrumentation, extensive method development and a proper combination FIGURE 2: Backpressures of columns with different FIGURE 7: Calculated peak capacities with columnextensive method development, and a proper combination of separation chemistries are necessary in this case The

FIGURE 2: Backpressures of columns with different t ti h hit t b t th

FIGURE 7: Calculated peak capacities with column chains in gradient mode: Hypersil GOLD 2 1 × 300 mmof separation chemistries are necessary in this case. The

theoretical basis has been summarized by Guiochon 1stationary phase architectures but the same chains in gradient mode: Hypersil GOLD 2.1 × 300 mm

l A 2 1 × 450 ltheoretical basis has been summarized by Guiochon.1 dimension of 2.1 × 100 mm. column or Accucore 2.1 × 450 mm column.

Highest resolution in shortest time can also be achieved Porous Particles d = 2 5 μm1000 700Highest resolution in shortest time can also be achieved by maximizing the peak-capacity-to-analysis-time ratio of

Porous Particles, dp 2.5 μmPorous Particles, dp= 1.9 μm F 0 45 0 65 0 60 0 45600

700

yby maximizing the peak-capacity-to-analysis-time ratio of an application This approach is easily implemented by

, p μSolid Core Particles, dp= 2.7 μm800) (mL/min)

0.45 0.65 0.60 0.45500600

city

an application. This approach is easily implemented by applying columns of extended length packed with porous

pSolid Core Particles, dp= 2.6 μmM lithi R dba

r)

tG 40 42 28 60500pa

c

applying columns of extended length packed with porous b 2 i ti l O th th h d h t

Monolithic Rod600e(b tG

(min)40 42 28 60400C

a

sub-2-micron particles. On the other hand, such a setup ure ( )

Pak

lead to a column head pressure up to 1200 bar and even 400ess P

(bar)930 1200 1200 850200P

e

more. In this work, experimental results from the use of Max Pressure of MonolithPr

e (bar)

n 430 600 480 670different stationary phase architectures in column lengths Max. Pressure of Monolith200

P nc 430 600 480 6700y p g

of 100 mm to 450 mm and at pressures up to 1200 bar are0

of 100 mm to 450 mm and at pressures up to 1200 bar are discussed Furthermore it is evaluated which approach 0 FIGURE 8: Ratio of peak-capacity-to-analysis-time with discussed. Furthermore, it is evaluated which approach provides the best peak-capacity-to-analysis-time ratio for

0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05Fl t F ( L/ i )

p p y ycolumn chains in gradient mode: Hypersil GOLD 2.1 ×provides the best peak-capacity-to-analysis-time ratio for

the analysis of complex sample mixturesFlow rate F (mL/min) g yp

300 mm column or Accucore 2.1 × 450 mm column.the analysis of complex sample mixtures. In one dimensional liquid chromatography the best peak

300 mm column or Accucore 2.1 450 mm column.17

M th dIn one-dimensional liquid chromatography, the best peak-

it t l i ti ti i hi d b l iti17

o FMethods capacity-to-analysis-time ratio is achieved by exploiting 14to

Rat

io F (mL/min)

0.45 0.65 0.60 0.45

Liquid Chromatography System the flow-pressure footprint of the system and column 11ci

ty

e R (mL/min)

/tLiquid Chromatography System™ ™ ™

hardware. High peak capacity is basically achieved 11

pac

Tim

e nc/tG( / )

11 14 17 11Thermo Scientific ™ Dionex™ UltiMate™ 3000 XRS system: through a high number of theoretical plates (N) per C

ap s T (1/min)

solvent rack SR-3000, pump LPG-3400XRS, sampler g g p ( ) p

column. The optimum linear velocity is determined by the 5ak C

lysi P 930 1200 1200 850p p p

WPS-3000TXRS, column thermostat TCC-3000RS, column. The optimum linear velocity is determined by the minimum theoretical plate height (Figure 1) Extending the P

eaA

nal

(bar)930 1200 1200 850

, ,and detector DAD-3000RS.

minimum theoretical plate height (Figure 1). Extending the column length means enhancing the column efficiency (N) 0

Aand detector DAD 3000RS. column length means enhancing the column efficiency (N) but also requires a higher pressure to achieve the same

0

Data Analysis but also requires a higher pressure to achieve the same linear velocity (Figure 2) The Accucore column chain provided higher peak capacities

Thermo Scientific™ Dionex™ Chromeleon™linear velocity (Figure 2). The Accucore column chain provided higher peak capacities

th th H il GOLD l h i t h fl tThermo Scientific Dionex ChromeleonChromatograph Data S stem Soft are ersion 6 8 Different concepts of stationary phase design were than the Hypersil GOLD column chain at each flow rate Chromatography Data System Software version 6.8 p y p g

compared by using identical column dimensions. The (Figure 7). The ratio between the peak capacities of both Comparison of 2 1x100 mm columns

p y gmonolithic rod showed the poorest efficiency but also column chains was the same as found under isocratic Comparison of 2.1x100 mm columns

C l Diff t d i f t ti h ( llmonolithic rod showed the poorest efficiency but also required the lowest flow-pressure footprint The maximum conditions by using phenones (Figure 5). But when

Columns: Different designs of stationary phase (all required the lowest flow-pressure footprint. The maximum operation pressure of a monolithic rod is about 200 bar

y g p ( g )comparing the ratio of peak capacity to analysis time, the

RP-C18) with 2.1 × 100 mm each operation pressure of a monolithic rod is about 200 bar and was almost reached for the 2 1 x 100 mm column at a

comparing the ratio of peak capacity to analysis time, the Accucore column chain generated 14 peaks per minute while

RP-5 Standard: Theophylline, p-nitroaniline, phenetole, and was almost reached for the 2.1 x 100 mm column at a fl t f 1 L/ i (Fi 2) H li

Accucore column chain generated 14 peaks per minute while the Hypersil GOLD column chain generated 17 peak per

o-xylene, and methyl benzoate flow rate of 1 mL/min (Figure 2). Hence, coupling li hi d h i hi h ld h l

the Hypersil GOLD column chain generated 17 peak per minute at 1200 bar backpressure (Figure 8) Thus byy , y

Mobile Phase A: Fisher Scientific™ Optima™ LC/MS water monolithic rods to a chain which would show an equal minute at 1200 bar backpressure (Figure 8). Thus, by exhausting the maximum pressure the Accucore chain wasMobile Phase A: Fisher Scientific Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrile number of theoretical plates compared to the other column exhausting the maximum pressure, the Accucore chain was bl t hi th hi h t k it b t th H ilMobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% B types is not feasible. able to achieve the highest peak capacity but the Hypersil OIsocratic Conditions: 50% A and 50% B

Flow Rate: 0 1 1 0 mL/minypHypersil GOLD and Accucore columns were found to

GOLD column chain provided the maximum ratio of peak Flow Rate: 0.1–1.0 mL/minInjection Volume:1 µL

Hypersil GOLD and Accucore columns were found to provide the highest column efficiency (Figure 1)

capacity to analysis time.Injection Volume:1 µLC l T t 30 °C

provide the highest column efficiency (Figure 1). F th th A l h d b t 60% fColumn Temperature: 30 °C Furthermore, the Accucore columns showed about 60% of th b k l ti t th H il GOLD l ConclusionDetection wavelength: 254 nm the backpressure relative to the Hypersil GOLD columns Conclusion

Detection Rate: 20 Hz at identical flow rates (Figure 2). Accordingly, to exploit the By exploiting the maximum pressure of the column Comparison of columns coupled in series flow-pressure footprint of the system and column

y p g phardware, a 2.1 × 450 mm long chain of Accucore C18Comparison of columns coupled in series

C l S i H il GOLD C18

p p yhardware, three Accucore 2.1 × 150 mm columns were

hardware, a 2.1 450 mm long chain of Accucore C18 (2 6 μm) columns could be built It produced a higherColumn-Series: Hypersil GOLD C18, ,

coupled in series to a sequence of 2.1 × 450 mm. For(2.6 μm) columns could be built. It produced a higher efficiency (N) than the 2 1 × 300 mm long Hypersil GOLD1.9 μm, 2.1 × 200 and 2.1 × 100 mm coupled in series to a sequence of 2.1 450 mm. For

Hypersil GOLD columns 2 1 × 200 mm and 2 1 × 100efficiency (N) than the 2.1 × 300 mm long Hypersil GOLD (1 9 μm) column chainAccucore C18, Hypersil GOLD columns , 2.1 × 200 mm and 2.1 × 100

mm were coupled to a sequence of 2 1 × 300 mm(1.9 μm) column chain.

2.6 μm, 2.1 × 150 mm (three) mm were coupled to a sequence of 2.1 × 300 mm. On the Accucore 2.1 × 450 mm column chain, a peakμ , ( )

Sample: Phenones and uracil Comparison of Columns Coupled in SeriesOn the Accucore 2.1 450 mm column chain, a peak capacity of 600 was achieved for the TCM “Dang GuiSample: Phenones and uracil

Mobile Phase A: Optima LC/MS waterp p

The series of Hypersil GOLD (2 1 × 300 mm) andcapacity of 600 was achieved for the TCM Dang Gui Long Hui Wan” with 42 min gradient time (14 peaks perMobile Phase A: Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrileThe series of Hypersil GOLD (2.1 × 300 mm) and Acc core (2 1 × 450 mm) col mns ere characteri ed

Long Hui Wan with 42 min gradient time (14 peaks per minute) A peak capacity of 670 could be realized with 60Mobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% BAccucore (2.1 × 450 mm) columns were characterized

ith lk l h t d d d i ti ditiminute). A peak capacity of 670 could be realized with 60

i di t ti hi h i d th k itIsocratic Conditions: 50% A and 50% BFlow Rate: 0 05 0 5 mL/min

with alkylphenone standard under isocratic conditions. min gradient time, which compromised the peak-capacity-t l i ti ti (11 k i t )Flow Rate: 0.05–0.5 mL/min

I j ti V l 5 L FIGURE 4: TheoreticalFIGURE 3 Theoretical to-analysis-time ratio (11 peaks per minute).Injection Volume: 5 μL FIGURE 4: Theoretical

plate numbers (N) ofFIGURE 3. Theoretical plate heights (H) of On the Hypersil GOLD 2 1 × 300 mm column chain aColumn Temperature: 35 °C plate numbers (N) of

hexanophenoneplate heights (H) of hexanophenone

On the Hypersil GOLD 2.1 × 300 mm column chain, a peak capacity of 430 was achieved for the TCM “DangDetection: 250 nm hexanophenone.hexanophenone. peak capacity of 430 was achieved for the TCM Dang G i L H i W “ i 40 i di t ti (11 kDetection Rate: 20 Hz N 100 x10³20m

)

Hypersil GOLD 2 1 x 300 mmGui Long Hui Wan“ in 40 min gradient time (11 peaks per

i t ) A k it f 480 d t i d ith 28T diti l Chi M di i (TCM) es 80

20

(µm

A 2 1 450

Hypersil GOLD 2.1 x 300 mm minute). A peak capacity of 480 was determined with 28 Traditional Chinese Medicine (TCM)

plat 80

16H ( Accucore 2.1 x 450 mm min gradient time, which benefited the peak-capacity-to-

Columns: see above

ofp

6016

ght analysis-time ratio (17 peaks per minute).

TCM-Sample: Dang Gui Long Hui Wan Nr. 3560 B, ero

4010Hei

g y ( p p )

M lithi l t id hi h k itiHerba Natura Harald Noll BV, Dinxperlo, the Netherlands mbe 4010

e H Monolithic columns cannot provide high peak capacities

i bl d h t ti d t th i i, p ,

Sample Preparation: 200 mg of sample mixed with 10 Num 204la

te in a comparable and short time due to their maximum Sample Preparation: 200 mg of sample mixed with 10 mL water sonicated for 15 min and filtered through

N 20

0 1 0 3 0 540 5 2 5 4 5

P operating pressure of 200 bar.mL water, sonicated for 15 min, and filtered through 0 2 μm PVDF membrane Flow rate F (mL/min)

0.1 0.3 0.50.5 2.5 4.5Linear Velocity u (mm/s)0.2 μm PVDF membrane

Mobile Phase A: 50 mM NH OAc pH 4 8Flow rate F (mL/min)Linear Velocity u (mm/s) ReferencesMobile Phase A: 50 mM NH4OAc, pH 4.8

Mobile Phase B: Optima LC/MS acetonitrile FIGURE 5 Peak capacities FIGURE 6: SystemReferences

1 G i h G Th Li it f th S ti P fMobile Phase B: Optima LC/MS acetonitrileG di t F 5% B t 50% B

FIGURE 5. Peak capacities (valid for all alkylphenones)

FIGURE 6: System backpressures

1. Guiochon, G. The Limits of the Separation Power of Gradient : From 5% B to 50% B, (valid for all alkylphenones). backpressures. Unidimensional Column liquid chromatography, Gradient Time: see figure (mentioned as tG)

1200J. Chromatogr. A, 2006, 1126, 6-49.

Flow Rate: see figure 220n c 1200) © 2014 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific IncInjection Volume: 2 μL ity ba

r © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe j μ

Column Temperature: 45 °C 180paci 800e(b the intellectual property rights of others.

PO71312-EN 0814SColumn Temperature: 45 CDetection wavelength: 280 nm C

ap sure

Detection wavelength: 280 nmDetection Rate: 50 Hz 140k

C

400ess

Detection Rate: 50 Hz 140

Pea 400

Pre

100

P

020 40 60 80R t ti Ti t ( i )

0 0.3 0.50.1Retention Time tR (min) Flow rate F (mL/min)

7Thermo Scientific Poster Note • PN71312-ISC-EN 0914S

Maximizing the Peak Capacity to Analysis Time Ratio in One DimensionalMaximizing the Peak-Capacity-to-Analysis-Time Ratio in One-Dimensional a g t e ea Capac ty to a ys s e at o O e e s o aCLiquid ChromatographyLiquid Chromatography

Mi h l H id S F b l d F k St iMichael Heidorn, Susanne Fabel and Frank SteinerMichael Heidorn, Susanne Fabel and Frank SteinerTh Fi h S i tifi G i GThermo Fisher Scientific, Germering, GermanyThermo Fisher Scientific, Germering, Germany

Overview Results While the theoretical plate heights (Figure 3) of both column-Overview Results p g ( g )series merged each other the same way as it was already

Purpose: A separation of complex sample mixtures with Comparison of 2 1 × 100 mm Columnsseries merged each other the same way as it was already seen in figure 1 the number of theoretical plates (Figure 4)Purpose: A separation of complex sample mixtures with

appropriate resolution in the shortest time possibleComparison of 2.1 × 100 mm Columns seen in figure 1, the number of theoretical plates (Figure 4)

was much higher for the longer sequence of Accucoreappropriate resolution in the shortest time possible. Different chromatographic columns (porous C18-silica, was much higher for the longer sequence of Accucore columns The maximum peak capacity of the AccucoreMethods: Different stationary phase architectures, column

g (fused-core silica-based C18, and monolithic silica-based columns. The maximum peak capacity of the Accucore

column chain was found to be n 220 at a retention time ofy p ,lengths, and temperatures, as well as applying pressures

,C18e) were tested by using the RP-5 standard under column chain was found to be nc = 220 at a retention time of

b t t 40 i (Fi 5) Th i k it flengths, and temperatures, as well as applying pressures up to 1200 bar

C18e) were tested by using the RP 5 standard under isocratic conditions (Figure 1 and Figure 2) about tR = 40 min (Figure 5). The maximum peak capacity of up to 1200 bar. isocratic conditions (Figure 1 and Figure 2). the Hypersil GOLD column was probably not achieved but

Results: Exploiting the maximum column pressure, the FIGURE 1: Efficiencies of columns with different found to be approximately nc = 150 at a retention time of p g p ,best peak-capacity-to-analysis-time ratio for isocratic stationary phase architectures but the same

y cabout tR = 25 min (Figure 5). The flow-pressure range was p p y y

conditions was determined by Thermo Scientific™y p

dimension of 2.1 × 100 mm.R ( g ) p g

similar for both column-series (Figure 6).conditions was determined by Thermo ScientificAccucore™ columns 2 6 μm particle size With gradient

dimension of 2.1 100 mm.Poro s Particles d 2 5 m C18 Comp Wm

)

similar for both column series (Figure 6).Accucore columns, 2.6 μm particle size. With gradient conditions Thermo Scientific™ Hypersil GOLD™ columns

Porous Particles, dp= 2.5 μm, C18, Comp. WPorous Particles d = 1 9 μm C18 Hypersil GOLD19(µ

m Traditional Chinese Medicine (TCM)conditions, Thermo Scientific Hypersil GOLD columns, 1 9 μm particle size were found to be superior

Porous Particles, dp= 1.9 μm, C18, Hypersil GOLDSolid Core Particles, dp= 2.6 μm, C18, Accucore17H

(

The series of Hypersil GOLD (2 1 × 300 mm) columns and1.9 μm particle size, were found to be superior. Solid Core Particles, dp 2.6 μm, C18, AccucoreSolid Core Particles, dp= 2.7 μm, C18, Comp. A15

17

ght The series of Hypersil GOLD (2.1 300 mm) columns and

Accucore (2 1 × 450 mm) columns were tested by using the

I t d tip μ p

Monolithic Rod, C18(e), Comp. M1315

eig

ne

Accucore (2.1 × 450 mm) columns were tested by using the TCM sample “Dang Gui Long Hui Wan“ under gradientIntroduction 13

e H

eyl

en TCM-sample Dang Gui Long Hui Wan under gradient conditions Applying the optimum linear velocity to each the

Th ti f ll l d ith i t11la

teo-

Xy conditions. Applying the optimum linear velocity to each, the th A l i i t d k itThe separation of all sample compounds with appropriate 9lP

lof

o three Accucore columns in series generated a peak capacity resolution in the shortest time possible is a common goal 7ic

al o of nc = 670 at the gradient time tg = 60 min with a of most chromatographers. This goal becomes even more 5re

ti gbackpressure of 850 bar. The two Hypersil GOLD columns in g g

relevant with highly complex samples. Generally, highest 35

eor

series generated a peak capacity of nc = 480 at the gradient g y p p y, gpeak capacities are achieved by employing an additional

30.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5Th

e g p p y c gtime tg = 28 min with a backpressure of 1200 bar.peak capacities are achieved by employing an additional

chromatographic dimension Dedicated instrumentation Linear Velocity u (mm/s)time tg 28 min with a backpressure of 1200 bar.

chromatographic dimension. Dedicated instrumentation, extensive method development and a proper combination FIGURE 2: Backpressures of columns with different FIGURE 7: Calculated peak capacities with columnextensive method development, and a proper combination of separation chemistries are necessary in this case The

FIGURE 2: Backpressures of columns with different t ti h hit t b t th

FIGURE 7: Calculated peak capacities with column chains in gradient mode: Hypersil GOLD 2 1 × 300 mmof separation chemistries are necessary in this case. The

theoretical basis has been summarized by Guiochon 1stationary phase architectures but the same chains in gradient mode: Hypersil GOLD 2.1 × 300 mm

l A 2 1 × 450 ltheoretical basis has been summarized by Guiochon.1 dimension of 2.1 × 100 mm. column or Accucore 2.1 × 450 mm column.

Highest resolution in shortest time can also be achieved Porous Particles d = 2 5 μm1000 700Highest resolution in shortest time can also be achieved by maximizing the peak-capacity-to-analysis-time ratio of

Porous Particles, dp 2.5 μmPorous Particles, dp= 1.9 μm F 0 45 0 65 0 60 0 45600

700

yby maximizing the peak-capacity-to-analysis-time ratio of an application This approach is easily implemented by

, p μSolid Core Particles, dp= 2.7 μm800) (mL/min)

0.45 0.65 0.60 0.45500600

city

an application. This approach is easily implemented by applying columns of extended length packed with porous

pSolid Core Particles, dp= 2.6 μmM lithi R dba

r)

tG 40 42 28 60500pa

c

applying columns of extended length packed with porous b 2 i ti l O th th h d h t

Monolithic Rod600e(b tG

(min)40 42 28 60400C

asub-2-micron particles. On the other hand, such a setup ur

e ( )

Pak

lead to a column head pressure up to 1200 bar and even 400ess P

(bar)930 1200 1200 850200P

e

more. In this work, experimental results from the use of Max Pressure of MonolithPr

e (bar)

n 430 600 480 670different stationary phase architectures in column lengths Max. Pressure of Monolith200

P nc 430 600 480 6700y p g

of 100 mm to 450 mm and at pressures up to 1200 bar are0

of 100 mm to 450 mm and at pressures up to 1200 bar are discussed Furthermore it is evaluated which approach 0 FIGURE 8: Ratio of peak-capacity-to-analysis-time with discussed. Furthermore, it is evaluated which approach provides the best peak-capacity-to-analysis-time ratio for

0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05Fl t F ( L/ i )

p p y ycolumn chains in gradient mode: Hypersil GOLD 2.1 ×provides the best peak-capacity-to-analysis-time ratio for

the analysis of complex sample mixturesFlow rate F (mL/min) g yp

300 mm column or Accucore 2.1 × 450 mm column.the analysis of complex sample mixtures. In one dimensional liquid chromatography the best peak

300 mm column or Accucore 2.1 450 mm column.17

M th dIn one-dimensional liquid chromatography, the best peak-

it t l i ti ti i hi d b l iti17

o FMethods capacity-to-analysis-time ratio is achieved by exploiting 14to

Rat

io F (mL/min)

0.45 0.65 0.60 0.45

Liquid Chromatography System the flow-pressure footprint of the system and column 11ci

ty

e R (mL/min)

/tLiquid Chromatography System™ ™ ™

hardware. High peak capacity is basically achieved 11

pac

Tim

e nc/tG( / )

11 14 17 11Thermo Scientific ™ Dionex™ UltiMate™ 3000 XRS system: through a high number of theoretical plates (N) per C

ap s T (1/min)

solvent rack SR-3000, pump LPG-3400XRS, sampler g g p ( ) p

column. The optimum linear velocity is determined by the 5ak C

lysi P 930 1200 1200 850p p p

WPS-3000TXRS, column thermostat TCC-3000RS, column. The optimum linear velocity is determined by the minimum theoretical plate height (Figure 1) Extending the P

eaA

nal

(bar)930 1200 1200 850

, ,and detector DAD-3000RS.

minimum theoretical plate height (Figure 1). Extending the column length means enhancing the column efficiency (N) 0

Aand detector DAD 3000RS. column length means enhancing the column efficiency (N) but also requires a higher pressure to achieve the same

0

Data Analysis but also requires a higher pressure to achieve the same linear velocity (Figure 2) The Accucore column chain provided higher peak capacities

Thermo Scientific™ Dionex™ Chromeleon™linear velocity (Figure 2). The Accucore column chain provided higher peak capacities

th th H il GOLD l h i t h fl tThermo Scientific Dionex ChromeleonChromatograph Data S stem Soft are ersion 6 8 Different concepts of stationary phase design were than the Hypersil GOLD column chain at each flow rate Chromatography Data System Software version 6.8 p y p g

compared by using identical column dimensions. The (Figure 7). The ratio between the peak capacities of both Comparison of 2 1x100 mm columns

p y gmonolithic rod showed the poorest efficiency but also column chains was the same as found under isocratic Comparison of 2.1x100 mm columns

C l Diff t d i f t ti h ( llmonolithic rod showed the poorest efficiency but also required the lowest flow-pressure footprint The maximum conditions by using phenones (Figure 5). But when

Columns: Different designs of stationary phase (all required the lowest flow-pressure footprint. The maximum operation pressure of a monolithic rod is about 200 bar

y g p ( g )comparing the ratio of peak capacity to analysis time, the

RP-C18) with 2.1 × 100 mm each operation pressure of a monolithic rod is about 200 bar and was almost reached for the 2 1 x 100 mm column at a

comparing the ratio of peak capacity to analysis time, the Accucore column chain generated 14 peaks per minute while

RP-5 Standard: Theophylline, p-nitroaniline, phenetole, and was almost reached for the 2.1 x 100 mm column at a fl t f 1 L/ i (Fi 2) H li

Accucore column chain generated 14 peaks per minute while the Hypersil GOLD column chain generated 17 peak per

o-xylene, and methyl benzoate flow rate of 1 mL/min (Figure 2). Hence, coupling li hi d h i hi h ld h l

the Hypersil GOLD column chain generated 17 peak per minute at 1200 bar backpressure (Figure 8) Thus byy , y

Mobile Phase A: Fisher Scientific™ Optima™ LC/MS water monolithic rods to a chain which would show an equal minute at 1200 bar backpressure (Figure 8). Thus, by exhausting the maximum pressure the Accucore chain wasMobile Phase A: Fisher Scientific Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrile number of theoretical plates compared to the other column exhausting the maximum pressure, the Accucore chain was bl t hi th hi h t k it b t th H ilMobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% B types is not feasible. able to achieve the highest peak capacity but the Hypersil OIsocratic Conditions: 50% A and 50% B

Flow Rate: 0 1 1 0 mL/minypHypersil GOLD and Accucore columns were found to

GOLD column chain provided the maximum ratio of peak Flow Rate: 0.1–1.0 mL/minInjection Volume:1 µL

Hypersil GOLD and Accucore columns were found to provide the highest column efficiency (Figure 1)

capacity to analysis time.Injection Volume:1 µLC l T t 30 °C

provide the highest column efficiency (Figure 1). F th th A l h d b t 60% fColumn Temperature: 30 °C Furthermore, the Accucore columns showed about 60% of th b k l ti t th H il GOLD l ConclusionDetection wavelength: 254 nm the backpressure relative to the Hypersil GOLD columns Conclusion

Detection Rate: 20 Hz at identical flow rates (Figure 2). Accordingly, to exploit the By exploiting the maximum pressure of the column Comparison of columns coupled in series flow-pressure footprint of the system and column

y p g phardware, a 2.1 × 450 mm long chain of Accucore C18Comparison of columns coupled in series

C l S i H il GOLD C18

p p yhardware, three Accucore 2.1 × 150 mm columns were

hardware, a 2.1 450 mm long chain of Accucore C18 (2 6 μm) columns could be built It produced a higherColumn-Series: Hypersil GOLD C18, ,

coupled in series to a sequence of 2.1 × 450 mm. For(2.6 μm) columns could be built. It produced a higher efficiency (N) than the 2 1 × 300 mm long Hypersil GOLD1.9 μm, 2.1 × 200 and 2.1 × 100 mm coupled in series to a sequence of 2.1 450 mm. For

Hypersil GOLD columns 2 1 × 200 mm and 2 1 × 100efficiency (N) than the 2.1 × 300 mm long Hypersil GOLD (1 9 μm) column chainAccucore C18, Hypersil GOLD columns , 2.1 × 200 mm and 2.1 × 100

mm were coupled to a sequence of 2 1 × 300 mm(1.9 μm) column chain.

2.6 μm, 2.1 × 150 mm (three) mm were coupled to a sequence of 2.1 × 300 mm. On the Accucore 2.1 × 450 mm column chain, a peakμ , ( )

Sample: Phenones and uracil Comparison of Columns Coupled in SeriesOn the Accucore 2.1 450 mm column chain, a peak capacity of 600 was achieved for the TCM “Dang GuiSample: Phenones and uracil

Mobile Phase A: Optima LC/MS waterp p

The series of Hypersil GOLD (2 1 × 300 mm) andcapacity of 600 was achieved for the TCM Dang Gui Long Hui Wan” with 42 min gradient time (14 peaks perMobile Phase A: Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrileThe series of Hypersil GOLD (2.1 × 300 mm) and Acc core (2 1 × 450 mm) col mns ere characteri ed

Long Hui Wan with 42 min gradient time (14 peaks per minute) A peak capacity of 670 could be realized with 60Mobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% BAccucore (2.1 × 450 mm) columns were characterized

ith lk l h t d d d i ti ditiminute). A peak capacity of 670 could be realized with 60

i di t ti hi h i d th k itIsocratic Conditions: 50% A and 50% BFlow Rate: 0 05 0 5 mL/min

with alkylphenone standard under isocratic conditions. min gradient time, which compromised the peak-capacity-t l i ti ti (11 k i t )Flow Rate: 0.05–0.5 mL/min

I j ti V l 5 L FIGURE 4: TheoreticalFIGURE 3 Theoretical to-analysis-time ratio (11 peaks per minute).Injection Volume: 5 μL FIGURE 4: Theoretical

plate numbers (N) ofFIGURE 3. Theoretical plate heights (H) of On the Hypersil GOLD 2 1 × 300 mm column chain aColumn Temperature: 35 °C plate numbers (N) of

hexanophenoneplate heights (H) of hexanophenone

On the Hypersil GOLD 2.1 × 300 mm column chain, a peak capacity of 430 was achieved for the TCM “DangDetection: 250 nm hexanophenone.hexanophenone. peak capacity of 430 was achieved for the TCM Dang G i L H i W “ i 40 i di t ti (11 kDetection Rate: 20 Hz N 100 x10³20m

)

Hypersil GOLD 2 1 x 300 mmGui Long Hui Wan“ in 40 min gradient time (11 peaks per

i t ) A k it f 480 d t i d ith 28T diti l Chi M di i (TCM) es 80

20

(µm

A 2 1 450

Hypersil GOLD 2.1 x 300 mm minute). A peak capacity of 480 was determined with 28 Traditional Chinese Medicine (TCM)

plat 80

16H ( Accucore 2.1 x 450 mm min gradient time, which benefited the peak-capacity-to-

Columns: see above

ofp

6016

ght analysis-time ratio (17 peaks per minute).

TCM-Sample: Dang Gui Long Hui Wan Nr. 3560 B, ero

4010Hei

g y ( p p )

M lithi l t id hi h k itiHerba Natura Harald Noll BV, Dinxperlo, the Netherlands mbe 4010

e H Monolithic columns cannot provide high peak capacities

i bl d h t ti d t th i i, p ,

Sample Preparation: 200 mg of sample mixed with 10 Num 204la

te in a comparable and short time due to their maximum Sample Preparation: 200 mg of sample mixed with 10 mL water sonicated for 15 min and filtered through

N 20

0 1 0 3 0 540 5 2 5 4 5

P operating pressure of 200 bar.mL water, sonicated for 15 min, and filtered through 0 2 μm PVDF membrane Flow rate F (mL/min)

0.1 0.3 0.50.5 2.5 4.5Linear Velocity u (mm/s)0.2 μm PVDF membrane

Mobile Phase A: 50 mM NH OAc pH 4 8Flow rate F (mL/min)Linear Velocity u (mm/s) ReferencesMobile Phase A: 50 mM NH4OAc, pH 4.8

Mobile Phase B: Optima LC/MS acetonitrile FIGURE 5 Peak capacities FIGURE 6: SystemReferences

1 G i h G Th Li it f th S ti P fMobile Phase B: Optima LC/MS acetonitrileG di t F 5% B t 50% B

FIGURE 5. Peak capacities (valid for all alkylphenones)

FIGURE 6: System backpressures

1. Guiochon, G. The Limits of the Separation Power of Gradient : From 5% B to 50% B, (valid for all alkylphenones). backpressures. Unidimensional Column liquid chromatography, Gradient Time: see figure (mentioned as tG)

1200J. Chromatogr. A, 2006, 1126, 6-49.

Flow Rate: see figure 220n c 1200) © 2014 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific IncInjection Volume: 2 μL ity ba

r © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe j μ

Column Temperature: 45 °C 180paci 800e(b the intellectual property rights of others.

PO71312-EN 0814SColumn Temperature: 45 CDetection wavelength: 280 nm C

ap sure

Detection wavelength: 280 nmDetection Rate: 50 Hz 140k

C

400ess

Detection Rate: 50 Hz 140

Pea 400

Pre

100

P

020 40 60 80R t ti Ti t ( i )

0 0.3 0.50.1Retention Time tR (min) Flow rate F (mL/min)

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PN71312-EN 0716S

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Maximizing the Peak Capacity to Analysis Time Ratio in One DimensionalMaximizing the Peak-Capacity-to-Analysis-Time Ratio in One-Dimensional a g t e ea Capac ty to a ys s e at o O e e s o aCLiquid ChromatographyLiquid Chromatography

Mi h l H id S F b l d F k St iMichael Heidorn, Susanne Fabel and Frank SteinerMichael Heidorn, Susanne Fabel and Frank SteinerTh Fi h S i tifi G i GThermo Fisher Scientific, Germering, GermanyThermo Fisher Scientific, Germering, Germany

Overview Results While the theoretical plate heights (Figure 3) of both column-Overview Results p g ( g )series merged each other the same way as it was already

Purpose: A separation of complex sample mixtures with Comparison of 2 1 × 100 mm Columnsseries merged each other the same way as it was already seen in figure 1 the number of theoretical plates (Figure 4)Purpose: A separation of complex sample mixtures with

appropriate resolution in the shortest time possibleComparison of 2.1 × 100 mm Columns seen in figure 1, the number of theoretical plates (Figure 4)

was much higher for the longer sequence of Accucoreappropriate resolution in the shortest time possible. Different chromatographic columns (porous C18-silica, was much higher for the longer sequence of Accucore columns The maximum peak capacity of the AccucoreMethods: Different stationary phase architectures, column

g (fused-core silica-based C18, and monolithic silica-based columns. The maximum peak capacity of the Accucore

column chain was found to be n 220 at a retention time ofy p ,lengths, and temperatures, as well as applying pressures

,C18e) were tested by using the RP-5 standard under column chain was found to be nc = 220 at a retention time of

b t t 40 i (Fi 5) Th i k it flengths, and temperatures, as well as applying pressures up to 1200 bar

C18e) were tested by using the RP 5 standard under isocratic conditions (Figure 1 and Figure 2) about tR = 40 min (Figure 5). The maximum peak capacity of up to 1200 bar. isocratic conditions (Figure 1 and Figure 2). the Hypersil GOLD column was probably not achieved but

Results: Exploiting the maximum column pressure, the FIGURE 1: Efficiencies of columns with different found to be approximately nc = 150 at a retention time of p g p ,best peak-capacity-to-analysis-time ratio for isocratic stationary phase architectures but the same

y cabout tR = 25 min (Figure 5). The flow-pressure range was p p y y

conditions was determined by Thermo Scientific™y p

dimension of 2.1 × 100 mm.R ( g ) p g

similar for both column-series (Figure 6).conditions was determined by Thermo ScientificAccucore™ columns 2 6 μm particle size With gradient

dimension of 2.1 100 mm.Poro s Particles d 2 5 m C18 Comp Wm

)

similar for both column series (Figure 6).Accucore columns, 2.6 μm particle size. With gradient conditions Thermo Scientific™ Hypersil GOLD™ columns

Porous Particles, dp= 2.5 μm, C18, Comp. WPorous Particles d = 1 9 μm C18 Hypersil GOLD19(µ

m Traditional Chinese Medicine (TCM)conditions, Thermo Scientific Hypersil GOLD columns, 1 9 μm particle size were found to be superior

Porous Particles, dp= 1.9 μm, C18, Hypersil GOLDSolid Core Particles, dp= 2.6 μm, C18, Accucore17H

(

The series of Hypersil GOLD (2 1 × 300 mm) columns and1.9 μm particle size, were found to be superior. Solid Core Particles, dp 2.6 μm, C18, AccucoreSolid Core Particles, dp= 2.7 μm, C18, Comp. A15

17

ght The series of Hypersil GOLD (2.1 300 mm) columns and

Accucore (2 1 × 450 mm) columns were tested by using the

I t d tip μ p

Monolithic Rod, C18(e), Comp. M1315

eig

ne

Accucore (2.1 × 450 mm) columns were tested by using the TCM sample “Dang Gui Long Hui Wan“ under gradientIntroduction 13

e H

eyl

en TCM-sample Dang Gui Long Hui Wan under gradient conditions Applying the optimum linear velocity to each the

Th ti f ll l d ith i t11la

teo-

Xy conditions. Applying the optimum linear velocity to each, the th A l i i t d k itThe separation of all sample compounds with appropriate 9lP

lof

o three Accucore columns in series generated a peak capacity resolution in the shortest time possible is a common goal 7ic

al o of nc = 670 at the gradient time tg = 60 min with a of most chromatographers. This goal becomes even more 5re

ti gbackpressure of 850 bar. The two Hypersil GOLD columns in g g

relevant with highly complex samples. Generally, highest 35

eor

series generated a peak capacity of nc = 480 at the gradient g y p p y, gpeak capacities are achieved by employing an additional

30.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5Th

e g p p y c gtime tg = 28 min with a backpressure of 1200 bar.peak capacities are achieved by employing an additional

chromatographic dimension Dedicated instrumentation Linear Velocity u (mm/s)time tg 28 min with a backpressure of 1200 bar.

chromatographic dimension. Dedicated instrumentation, extensive method development and a proper combination FIGURE 2: Backpressures of columns with different FIGURE 7: Calculated peak capacities with columnextensive method development, and a proper combination of separation chemistries are necessary in this case The

FIGURE 2: Backpressures of columns with differentt ti h hit t b t th

FIGURE 7: Calculated peak capacities with column chains in gradient mode: Hypersil GOLD 2 1 × 300 mmof separation chemistries are necessary in this case. The

theoretical basis has been summarized by Guiochon 1stationary phase architectures but the same chains in gradient mode: Hypersil GOLD 2.1 × 300 mm

l A 2 1 × 450 ltheoretical basis has been summarized by Guiochon.1 dimension of 2.1 × 100 mm. column or Accucore 2.1 × 450 mm column.

Highest resolution in shortest time can also be achieved Porous Particles d = 2 5 μm1000 700Highest resolution in shortest time can also be achievedby maximizing the peak-capacity-to-analysis-time ratio of

Porous Particles, dp 2.5 μmPorous Particles, dp= 1.9 μm F 0 45 0 65 0 60 0 45600

700

yby maximizing the peak-capacity-to-analysis-time ratio ofan application This approach is easily implemented by

, p μSolid Core Particles, dp= 2.7 μm800) (mL/min)

0.45 0.65 0.60 0.45500600

city

an application. This approach is easily implemented by applying columns of extended length packed with porous

pSolid Core Particles, dp= 2.6 μmM lithi R dba

r)

tG 40 42 28 60500pa

c

applying columns of extended length packed with porous b 2 i ti l O th th h d h t

Monolithic Rod600e(b tG

(min)40 42 28 60400C

a

sub-2-micron particles. On the other hand, such a setup ure ( )

Pak

lead to a column head pressure up to 1200 bar and even 400ess P

(bar)930 1200 1200 850200P

e

more. In this work, experimental results from the use ofMax Pressure of MonolithPr

e (bar)

n 430 600 480 670different stationary phase architectures in column lengths Max. Pressure of Monolith200

P nc 430 600 480 6700y p g

of 100 mm to 450 mm and at pressures up to 1200 bar are0

of 100 mm to 450 mm and at pressures up to 1200 bar are discussed Furthermore it is evaluated which approach 0 FIGURE 8: Ratio of peak-capacity-to-analysis-time with discussed. Furthermore, it is evaluated which approach provides the best peak-capacity-to-analysis-time ratio for

0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05Fl t F ( L/ i )

p p y ycolumn chains in gradient mode: Hypersil GOLD 2.1 ×provides the best peak-capacity-to-analysis-time ratio for

the analysis of complex sample mixturesFlow rate F (mL/min) g yp

300 mm column or Accucore 2.1 × 450 mm column.the analysis of complex sample mixtures. In one dimensional liquid chromatography the best peak

300 mm column or Accucore 2.1 450 mm column.17

M th dIn one-dimensional liquid chromatography, the best peak-

it t l i ti ti i hi d b l iti17

o FMethods capacity-to-analysis-time ratio is achieved by exploiting 14to

Rat

io F (mL/min)

0.45 0.65 0.60 0.45

Liquid Chromatography System the flow-pressure footprint of the system and column 11ci

ty

e R (mL/min)

/tLiquid Chromatography System™ ™ ™

hardware. High peak capacity is basically achieved 11

pac

Tim

e nc/tG( / )

11 14 17 11Thermo Scientific ™ Dionex™ UltiMate™ 3000 XRS system: through a high number of theoretical plates (N) per C

ap sT (1/min)

solvent rack SR-3000, pump LPG-3400XRS, samplerg g p ( ) p

column. The optimum linear velocity is determined by the 5ak C

lysi P 930 1200 1200 850p p p

WPS-3000TXRS, column thermostat TCC-3000RS, column. The optimum linear velocity is determined by the minimum theoretical plate height (Figure 1) Extending the P

eaA

nal

(bar)930 1200 1200 850

, ,and detector DAD-3000RS.

minimum theoretical plate height (Figure 1). Extending the column length means enhancing the column efficiency (N) 0

Aand detector DAD 3000RS. column length means enhancing the column efficiency (N) but also requires a higher pressure to achieve the same

0

Data Analysis but also requires a higher pressure to achieve the same linear velocity (Figure 2) The Accucore column chain provided higher peak capacities

Thermo Scientific™ Dionex™ Chromeleon™linear velocity (Figure 2). The Accucore column chain provided higher peak capacities

th th H il GOLD l h i t h fl tThermo Scientific Dionex ChromeleonChromatograph Data S stem Soft are ersion 6 8 Different concepts of stationary phase design were than the Hypersil GOLD column chain at each flow rate Chromatography Data System Software version 6.8 p y p g

compared by using identical column dimensions. The (Figure 7). The ratio between the peak capacities of both Comparison of 2 1x100 mm columns

p y gmonolithic rod showed the poorest efficiency but also column chains was the same as found under isocratic Comparison of 2.1x100 mm columns

C l Diff t d i f t ti h ( llmonolithic rod showed the poorest efficiency but also required the lowest flow-pressure footprint The maximum conditions by using phenones (Figure 5). But when

Columns: Different designs of stationary phase (all required the lowest flow-pressure footprint. The maximum operation pressure of a monolithic rod is about 200 bar

y g p ( g )comparing the ratio of peak capacity to analysis time, the

RP-C18) with 2.1 × 100 mm each operation pressure of a monolithic rod is about 200 bar and was almost reached for the 2 1 x 100 mm column at a

comparing the ratio of peak capacity to analysis time, the Accucore column chain generated 14 peaks per minute while

RP-5 Standard: Theophylline, p-nitroaniline, phenetole, and was almost reached for the 2.1 x 100 mm column at a fl t f 1 L/ i (Fi 2) H li

Accucore column chain generated 14 peaks per minute while the Hypersil GOLD column chain generated 17 peak per

o-xylene, and methyl benzoate flow rate of 1 mL/min (Figure 2). Hence, couplingli hi d h i hi h ld h l

the Hypersil GOLD column chain generated 17 peak per minute at 1200 bar backpressure (Figure 8) Thus byy , y

Mobile Phase A: Fisher Scientific™ Optima™ LC/MS water monolithic rods to a chain which would show an equal minute at 1200 bar backpressure (Figure 8). Thus, by exhausting the maximum pressure the Accucore chain wasMobile Phase A: Fisher Scientific Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrile number of theoretical plates compared to the other column exhausting the maximum pressure, the Accucore chain was bl t hi th hi h t k it b t th H ilMobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% B types is not feasible. able to achieve the highest peak capacity but the Hypersil OIsocratic Conditions: 50% A and 50% B

Flow Rate: 0 1 1 0 mL/minypHypersil GOLD and Accucore columns were found to

GOLD column chain provided the maximum ratio of peak Flow Rate: 0.1–1.0 mL/minInjection Volume:1 µL

Hypersil GOLD and Accucore columns were found to provide the highest column efficiency (Figure 1)

capacity to analysis time.Injection Volume:1 µLC l T t 30 °C

provide the highest column efficiency (Figure 1). F th th A l h d b t 60% fColumn Temperature: 30 °C Furthermore, the Accucore columns showed about 60% of th b k l ti t th H il GOLD l ConclusionDetection wavelength: 254 nm the backpressure relative to the Hypersil GOLD columns Conclusion

Detection Rate: 20 Hz at identical flow rates (Figure 2). Accordingly, to exploit the By exploiting the maximum pressure of the column Comparison of columns coupled in series flow-pressure footprint of the system and column

y p g phardware, a 2.1 × 450 mm long chain of Accucore C18Comparison of columns coupled in series

C l S i H il GOLD C18

p p yhardware, three Accucore 2.1 × 150 mm columns were

hardware, a 2.1 450 mm long chain of Accucore C18 (2 6 μm) columns could be built It produced a higherColumn-Series: Hypersil GOLD C18, ,

coupled in series to a sequence of 2.1 × 450 mm. For(2.6 μm) columns could be built. It produced a higher efficiency (N) than the 2 1 × 300 mm long Hypersil GOLD1.9 μm, 2.1 × 200 and 2.1 × 100 mm coupled in series to a sequence of 2.1 450 mm. For

Hypersil GOLD columns 2 1 × 200 mm and 2 1 × 100efficiency (N) than the 2.1 × 300 mm long Hypersil GOLD (1 9 μm) column chainAccucore C18, Hypersil GOLD columns , 2.1 × 200 mm and 2.1 × 100

mm were coupled to a sequence of 2 1 × 300 mm(1.9 μm) column chain.

2.6 μm, 2.1 × 150 mm (three) mm were coupled to a sequence of 2.1 × 300 mm. On the Accucore 2.1 × 450 mm column chain, a peakμ , ( )

Sample: Phenones and uracil Comparison of Columns Coupled in SeriesOn the Accucore 2.1 450 mm column chain, a peakcapacity of 600 was achieved for the TCM “Dang GuiSample: Phenones and uracil

Mobile Phase A: Optima LC/MS waterp p

The series of Hypersil GOLD (2 1 × 300 mm) andcapacity of 600 was achieved for the TCM Dang GuiLong Hui Wan” with 42 min gradient time (14 peaks perMobile Phase A: Optima LC/MS water

Mobile Phase B: Optima LC/MS acetonitrileThe series of Hypersil GOLD (2.1 × 300 mm) and Acc core (2 1 × 450 mm) col mns ere characteri ed

Long Hui Wan with 42 min gradient time (14 peaks per minute) A peak capacity of 670 could be realized with 60Mobile Phase B: Optima LC/MS acetonitrile

Isocratic Conditions: 50% A and 50% BAccucore (2.1 × 450 mm) columns were characterized

ith lk l h t d d d i ti ditiminute). A peak capacity of 670 could be realized with 60

i di t ti hi h i d th k itIsocratic Conditions: 50% A and 50% BFlow Rate: 0 05 0 5 mL/min

with alkylphenone standard under isocratic conditions. min gradient time, which compromised the peak-capacity-t l i ti ti (11 k i t )Flow Rate: 0.05–0.5 mL/min

I j ti V l 5 L FIGURE 4: TheoreticalFIGURE 3 Theoretical to-analysis-time ratio (11 peaks per minute).Injection Volume: 5 μL FIGURE 4: Theoretical

plate numbers (N) ofFIGURE 3. Theoretical plate heights (H) of On the Hypersil GOLD 2 1 × 300 mm column chain aColumn Temperature: 35 °C plate numbers (N) of

hexanophenoneplate heights (H) of hexanophenone

On the Hypersil GOLD 2.1 × 300 mm column chain, apeak capacity of 430 was achieved for the TCM “DangDetection: 250 nm hexanophenone.hexanophenone. peak capacity of 430 was achieved for the TCM Dang G i L H i W “ i 40 i di t ti (11 kDetection Rate: 20 Hz N 100 x10³20m

)

Hypersil GOLD 2 1 x 300 mmGui Long Hui Wan“ in 40 min gradient time (11 peaks per

i t ) A k it f 480 d t i d ith 28T diti l Chi M di i (TCM) es 80

20

(µm

A 2 1 450

Hypersil GOLD 2.1 x 300 mm minute). A peak capacity of 480 was determined with 28 Traditional Chinese Medicine (TCM)

plat 80

16H( Accucore 2.1 x 450 mm min gradient time, which benefited the peak-capacity-to-

Columns: see above

ofp

6016

ght analysis-time ratio (17 peaks per minute).

TCM-Sample: Dang Gui Long Hui Wan Nr. 3560 B, ero

4010Hei

g y ( p p )

M lithi l t id hi h k itiHerba Natura Harald Noll BV, Dinxperlo, the Netherlands mbe 4010

e H Monolithic columns cannot provide high peak capacities

i bl d h t ti d t th i i, p ,

Sample Preparation: 200 mg of sample mixed with 10 Num 204la

te in a comparable and short time due to their maximum Sample Preparation: 200 mg of sample mixed with 10 mL water sonicated for 15 min and filtered through

N 20

0 1 0 3 0 540 5 2 5 4 5

P operating pressure of 200 bar.mL water, sonicated for 15 min, and filtered through 0 2 μm PVDF membrane Flow rate F (mL/min)

0.1 0.3 0.50.5 2.5 4.5Linear Velocity u (mm/s)0.2 μm PVDF membrane

Mobile Phase A: 50 mM NH OAc pH 4 8Flow rate F (mL/min)Linear Velocity u (mm/s) ReferencesMobile Phase A: 50 mM NH4OAc, pH 4.8

Mobile Phase B: Optima LC/MS acetonitrile FIGURE 5 Peak capacities FIGURE 6: SystemReferences

1 G i h G Th Li it f th S ti P fMobile Phase B: Optima LC/MS acetonitrileG di t F 5% B t 50% B

FIGURE 5. Peak capacities (valid for all alkylphenones)

FIGURE 6: Systembackpressures

1. Guiochon, G. The Limits of the Separation Power ofGradient : From 5% B to 50% B, (valid for all alkylphenones). backpressures. Unidimensional Column liquid chromatography, Gradient Time: see figure (mentioned as tG)

1200J. Chromatogr. A, 2006, 1126, 6-49.

Flow Rate: see figure 220n c 1200) © 2014 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific IncInjection Volume: 2 μL ity ba

r © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe j μ

Column Temperature: 45 °C 180paci 800e(b the intellectual property rights of others.

PO71312-EN 0814SColumn Temperature: 45 CDetection wavelength: 280 nm C

ap sure

Detection wavelength: 280 nmDetection Rate: 50 Hz 140k

C

400ess

Detection Rate: 50 Hz 140

Pea 400

Pre

100

P

020 40 60 80R t ti Ti t ( i )

0 0.3 0.50.1Retention Time tR (min) Flow rate F (mL/min)