implementation of high resolution fast lc · pdf file · 2015-07-28power n(5...

Implementation of

High Resolution Fast LC

Great idea but what will I have to change?

Agilent Technologies 2012

High Performance Series

How Difficult is Implementation?

Depends on Your Separation Goal

• Simple 2x-3x Speed Improvement – Easy

• 2x-3x Improvement in Resolution – Moderate

• 5x + Speed Improvement – More Involved

What Method Parameters Are Important?

Method

Parameter

Isocratic Speed

Gradient Speed

Flow Rate

Gradient Time

Pressure

Temperature

Detection

Dwell Volume

What Is Your Current Column Efficiency and

How Fast Do You Want To Run?

Column

Length (mm)

Resolving

Power

N(5 µm)

Resolving

Power

N(3.5 µm)

Resolving

Power

N(1.8 µm)

Typical

Pressure

Bar (1.8 µm)

150 12,500 21,000 32,500 560

100 8,500 14,000 24,000 420

75 6000 10,500 17,000 320

50 4,200 7,000 12,000 210

30 N.A. 4,200 6,500 126

15 N.A. 2,100 2,500 55

Analysis Time

Peak Volume

Analysis

Time*

-33%

-50%

-67%

-80%

-90%

Solvent Usage

* Reduction in analysis time compared to 150 mm column

• pressure determined with 60:40 MeOH/water, 1ml/min, 4.6mm ID

“RULE OF THUMB”

• Set of Approximations based on chromatographic

behavior and mathematical relationships

• Will deliver nearly the desired goal

• Probably need to be tweaked to deliver best results

Simple Isocratic Method Transfer for Speed

Step 1

• Reduce Column Length and Particle Size

• Maintain Flow Rate

Step 2 Faster

• Increase Flow Rate

• Pressure too High? Increase Temperature to Lower Pressure

• Increase Flow Rate Again

Step 3 Even Faster (Need Instrument Optimization)

• Decrease Column Diameter (2.1mm), Reduce Flow Rate Proportionately

• Reduce Injection Volume Based on Ratio of Column Volumes

•When flow limit of pump is reached

•When approaching about 80 - 90% pressure

limit of instrument

•When resolution is no longer satisfactory

When to Stop!?

Reduce Column Length/Particle Size by Same Ratio

5um 1.8um

Reduce column length by factor of 3

3.5 um 1.8um

Reduce column length by factor of 2

Reduced Column Length/Particle Size

No Instrument Changes - Easy

Columns: Eclipse Plus C18, as described below. Mobile Phase: A: water, B: MeOH, (15:85) Injection volume: 6uL

Temperature: 25°C Flow: 1 mL/min. Detection: 310, 4 nm, 0.5 s response time, semi-micro flow cell, Sample: Sunscreens

min 0 2 4 6 8 10 12 14

mAU

0

20

40

60

min 0 2 4 6 8 10 12 14

mAU

0

20

40

60

80

100

min 0 2 4 6 8 10 12 14

mAU

0

50

100

150

4.6 x 100 mm, 3.5 µm

P=105 bar

1

2 3

4

Rs3,2= 6.65

Rs3,2= 6.51

Rs3,2= 6.41

4.6 x 50 mm, 1.8 µm

P=208 bar

4.6 x 150 mm, 5 µm

P=82 bar

Reached Flow Rate Limit? Reduce Column I.D./Flow

4.6 mm 3 mm

Reduce flow rate by factor of 0.4

2.1 mm 4.6 mm

Reduce flow rate by factor of 0.2

Flow Modification – 4.6mm to 2.1mm I.D. Column

ml/minmm

mmml/min 21.0

2.30

1.051.0 i.e.

2

2 col.

2

column1

column21 col. Flow

Radius

RadiusFlow

Reducing Column Size? Reduce Injection Volume!

2 col.

column1

column21 col. Inj.Vol.

Volume

VolumeInj.Vol.

2 col.column1

column21 col. 4

2.0

4.020 i.e. μl

ml

mlμl

Zorbax column volume = 3.14 x r2 x L x 0.6 (r and L in cm) column2Radius

Reduce injection volume

4.6 mm 3 mm

Reduction to allow for diameter change

2.1 mm 4.6 mm

= 0.4 x Original

= 0.2 x Original

x Reduction to allow for length change

150 mm 50 mm = 0.33 x Original

Example - 4.6mm x 150mm transferred to 2.1mm x 100mm

= 0.2 x 0.67

=0.13 x original injection volume



0 10 20 30 40

Relationship of k* and Key Gradient Parameters

Time (min)

100% B

100% B

100% B

100% B

tg=

40

tg=

20

tg=

10

tg=

5

tg F

S DF Vm

k*

F = flow rate (mL/min.) tg = gradient time (min.) Vm = column volume (mL) DF = % B change S = constant

0% B

0% B

0% B

0% B

Simplified Gradient Method Transfer

Step 1

• Reduce Column Length and Particle Size

• Adjust Gradient Time by same Factor

• Maintain Flow Rate

Step 2 Faster

• Increase Flow and Reduce Gradient Time

• Stop When Reach Flow Limits of Instrument

Step 3 Even Faster

• Decrease Diameter of Column (2.1mm)

• Match Flow to New Column Diameter

• Reduce Injection Volume

• Repeat Step 2 Until Reach 80-90 % Instrument Pressure Limit

Example of Possible Speed Increase

F= 1.20ml/min

T = 40°C

Analysis Time = 11min

4.6mm x 150mm 5.0µm

min 0 2 4 6 8 10 12

F = 4.80ml/min

T = 40°C

Analysis Time = 1.05min

4.6mm x 50mm 5.0µm

min 0 0.2 0.4 0.6 0.8 1

F= 1.00ml/min

T = 40°C

Analysis Time = 1.1min

2.1mm x 50mm 1.8µm

min 0.2 0.4 0.6 0.8 1 0

Max Speed at T = 95oC

2.1mm x 50mm 1.8um

F= 2.40ml/min

T = 95°C

Analysis Time: 0.4min

PWHH = 197msec

min 0.2 0.4 0.6 0.8 1 0

> 20x faster !

Optimizing Gradient Separations with 1.8 um RRHT

Columns: 10 X Faster Analysis

min 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25

min 5 10 15 20 25

RRHT SB-C18

2.1 x 50mm, 1.8um

Temp: 50°C

Flow: 1 mL/min

Gradient (tG): 2.4 min

Rapid Resolution SB-C18

3.0 x 150mm, 3.5um

Temp: 25°C

Flow: 1.4 mL/min

Gradient (tG) : 18 min

SB-C18

4.6 x 250mm, 5um

Temp: 25°C

Flow: 1mL/min

Gradient (tG): 30 min

0 2 4 6 8 10

12

.

• Flow rate vs. Gradient time vs.

Peak capacity

• For small molecules(MW < ~1000)

• Different Column Lengths

• Broken lines are isobar (800 bar)

What Length Column Yields Highest Peak Capacity?

50mm

150mm

100mm

Shorter Columns with Fast Gradients

Yield Higher Peak Capacity

50mm

150mm

100mm

Shorter Gradient (5 min)

Peak Capacity:

• 258 for 50 mm

• 240 for 100 mm

• 221 for 150 mm

Separation of 12 Phenols on Poroshell 120 EC-C18

5 minutes – 50mm Column

274 bar 2.5 ml/min

min 0 5

Conditions:

Column: Poroshell 120 EC-C18, 4.6 x 50mm, 2.7um

Mobile Phase:

Solvent A: Water with 0.1% Formic Acid

Solvent B: Acetonitrile

Gradient::

Time %B

0.8 5%

6.8 60%

1200 SL controlled temperature at 25 C

2 mm flow cell

1. Hydroquinone

2. Resourcinol

3. Catechol

4. Phenol

5. 4-Nitrophenol

6. p-cresol

7. o-cresol

8. 2-Nitrophenol

9. 3,4 di methyl phenol



12. 1-napthol

Poroshell 120 gives high efficiency, high resolution separations quickly at HPLC pressures.

Long Gradient (40 min)

Peak Capacity:

• 422 for 50 mm

• 510 for 100 mm

• 525 for 150 mm

Longer Columns with Long Gradient Times Yield

Greater Peak Capacity

50mm

150mm

100mm

Constant Particle Size, Gradient Time More Resolution with Longer Column

Group/Presentation Title

Agilent Restricted

Month ##, 200X Page 22

min 0 2 4 6 8 10 12 14 16 18

mAU

0

50

100

150

200

min 0 2 4 6 8 10 12 14 16 18

mAU

0

50

100

150

200

min 0 2 4 6 8 10 12 14 16 18

mAU

0

50

100

150

200

RRHD SB-C18 2.1 x 50 mm, 1.8um

Pmax=366 bar

nc = 424

RRHD SB-C18 2.1 x 100 mm , 1.8um

Pmax=595 bar

nc = 485

RRHD SB-C18 2.1 x 150 mm, 1.8um

Pmax=768 bar

nc = 589

Rs: 2.40

Rs: 1.37

Rs: 0

HPLC Instrument Components

Gradient Delay or Dwell Volume .

Extracolumn Volume

Data Sampling or Acquistion Rate

.

Number of

Scans or points

Minor Dwell Volume Differences

Can Change Resolution

0 10 20 30 40

0 10 20 30 40

VD = 0.43

mL

Column: ZORBAX Rapid Resolution Eclipse

XDB-C8

4.6 x 75 mm, 3.5 µm

Mobile Phase: Gradient, 0 - 100 %B in 52.5

min.

A: 5/95 methanol/ 25 mM phosphate

pH 2.50

B: 80/20 methanol/25 mM phosphate

pH 2.50

Flow Rate: 0.5 mL/min

Temperature: 25°C

Injection: 5 L

Detection: 250 nm

Sample: Mixture of antibiotics and antidepressants

Upper trace simulates actual run data

entered into DryLab® 3.0 software

Lower trace is simulated chromatogram for

larger VD

VD = 2.0

mL

1100/1200 Configurations for Cost Effective Fast

and Ultra-Fast HPLC

* Pieces to upgrade, in kits

High pressure

Gradient pump

Std or Well

Plate sampler

Diode Array

Detector

Standard assembly

without standard mixer

0.12 x 400 mm capillary

DAD equipped with a 1.7

L flow cell

Mass

Spectrometer

0.12 x XX mm PEEK Capillary

High pressure

Gradient pump

Std or Well

Plate sampler

Rapid Resolution

HT Column

Diode Array

detector

Waste




DAD equipped with a 5uL

or 1.7 L flow cell

3 L

heat exchanger

Thermostatted

Column

compartment

4.6 mm ID columns 2.1 mm ID columns

Cell Inlet Capillary

Cell Outlet Capillary

Rapid Resolution

HT Column



3 L

heat exchanger

Thermostatted

Column

compartment

Cell Inlet Capillary

Cell Outlet Capillary

Why Optimize System Volume?

min 0.5 1 1.5 2 2.5

mAU

0

100

200

350

400

550

600 System Tubing Volume Optimized

0.12mm i.d. tubing

Peak width 0.018 min


Resolution 1.902

min 0.5 1 1.5 2 2.5

mAU

0

100

200

300

400

System Tubing Volume Not Optimized

0.17mm i.d. tubing



Resolution 0.961


Agilent Restricted

Month ##, 200X June, 2011

Use 1200bar UHPLC for Best Speed ZORBAX SB-C18 2.1 x 50mm 1.8 µm

June 06, 2005 Page 27

F = 2 ml/min

P = 975 bar

F = 2.3 ml/min

P = 1110 bar

0.5min

0.4min

min 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

mAU

0

100

200

300

400

500

min 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

mAU

0

100

200

300

400

500

Eclipse Plus C18 2.1 mm × 100 mm, 1.8 μm column at a flow rate of 0.5 mL/min.

A = 5 mM acetic acid in water B = 100% acetonitrile, Gr= 5-95% B

300 Pesticides < 20 minutes, 1290 Infinity

Ultrafast LC/MS Analysis for 15 Analyte Subset

1290 Infinity Applications

Peak Width 0.7 sec

RRHD Eclipse Plus C18

2.1x 50 mm, 1.8 um

750 bar

1 minute

Time Composition

0.0 10% ACN

1.5 100% ACN

Ultimate speed on a short column with ballistic gradient

Difference in Extra-Column Volume and

Performance

Default 1290

Total Extra-Column Volume:

• 3.8+2.5+2.3=8.6 µL

Optimized 1290

Total Extra Column Volume:

• 1.1+1.1+0.8=3 µL

Effect of Extra-column Volume on a Gradient

Analysis of Alkylphenones

Default 1290, 8.6 µL Extra-column Volume

Optimized 1290, 3.0 µL Extra-column Volume

Pmax=320 bar

Rs5,6=1.18

nC=35

Pmax=323 bar

Rs5,6=2.25 +91%

nC=56 +60%

What Happens If the Connections Poorly Made ?

If Dimension X is too long, leaks will occur

Ferrule cannot seat properly

Mixing Chamber

If Dimension X is too short, a dead-volume,

or mixing chamber, will occur

Wrong … too long

Wrong … too

short X

X

min 0 0.1 0.2 0.3 0.4

mAU

0

20

40

60

80

100

120

140 One bad capillary connection!

min 0 0.1 0.2 0.3 0.4

mAU

0

30

60

90

120

150

180

210 Fixed!

130 mAU

160 mAU

Influence of Bad Post-Column Connection

Effect of Data Acquisition Rate (time constant) Peak Width, Resolution and Peak Capacity in Ultra-Fast LC

min 0.1 0.2 0.3 0.4 0.5 0

80Hz

PW=0.30sec

40Hz

PW = 0.33 sec

20 Hz

PW=0.42sec

10Hz

PW=0.67sec

5Hz PW=1.24sec

•

80Hz vvsvs.us 20Hz

– 30% Peak Width

+ 30% Resolution

+ 40% Peak Capacity

+ 70% Apparent Column Efficiency

80Hz vervs.s 10Hz

– 55% Peak Width

+ 90% Resolution

+ 120% Peak Capacity

+ 260% Apparent Column Efficiency

Poroshell 120 Resists Plugging with 2 um Frit

Challenging Samples - Plasma

35

Diflusinal in Plasma

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

360

380

400

1 501 1001 1501 2001 2501

Injections

Pre

ssu

re

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

Eff

icie

ncy (

N)

End Press

Plates

Column: Poroshell 120 EC-C18, 3.0 x 50mm, 2.7um LC: Agilent 1200 RRLC (SL)

Sample: Precipitated Plasma: 2 parts Plasma: 7 Parts 20/80 Water-MeCN w/0.1 % Formic Acid with 1 Part Diflusinal

in 50/50 Water-MeCN 10 ug/ml (Final concentration Diflusinal 1 ug/ml) Shaken and allowed to settle 10 minutes

Not Centrifuged/ Not Filtered

Injection Volume: 1ul injections

Solvent A: Water w/0.1 % TFA

Solvent B: MeCN w/0.08 % TFA

Flow Rate 1 ml/min 1 ul injection

Time % B

0 20

0.5 90

0.6 90

1.1 20

2.5 20

Why Filter the Sample?

Extreme Performance Requires Better Sample “Hygiene”

• Prevents blocking of capillaries, frits, and the column inlet

• Results in less wear and tear on the critical moving parts of injection valves

• Results in less downtime of the instrument for repairs

• Produces improved analytical results by removing potentially interfering contamination

In-Line Filters Provide Good Insurance

Against System OverPressure

Summary

• Poroshell 120 Columns will improve performance of old LCs

• 2x-3X speed increase needs little more than a column change

• Same I.D., shorter Column, smaller Particle

• 5x+ Speed or Sensitivity May Require a 2.1mm I.D. Column

• 2.1mm will require optimizing extra-column volume in most instruments

• Kits are available to improve instrument performance

• Maximum performance will be realized on new design LCs

• Very complex samples will need UHPLC instruments and columns

APPENDIX

A Tip for Controlling Unnecessary Pressure

A Bit of Attention to Filtering Might Be a Good Idea!

Protect HPLC Systems From Premature Wear and Over

Pressure Shutdown by Using Effective Filtration

•Filter Buffers

•Filter Samples

•Use Mobile Phase Miscible Sample Solvents

•Use Pre-Column Filters

RRHT Column Installation Recommendations to

Avoid Complaints of High Pressure

1. Purge the pumps (connections up to the column) of any buffer containing mobile phases. Flush through 5 mL of solvent before attaching the column to instrument.

2. Flush the column with compatible mobile phase (compatible with the solvents the column was shipped in) starting slowly at 0.1 mL/min for a 2.1 mm ID column, 0.2 mL/min for a 3.0 mm ID column, and 0.4 mL/min for 4.6 mm ID. This is done because when the new mobile phase reaches the column a spike in pressure will occur when the different solvents mix. The low flow rate allows this to happen without causing overpressuring on the LC system. Increase the flow rate to the desired flow over 5 minutes.

3. Once the pressure has stabilized, attach the column to the detector.

4. Equilibrate the column and detector with 10 column volumes of the mobile phase prior to use.

5. If you are running a gradient, check that the pressure range of the gradient – which may be 100 – 130 bar or more, will not cause the system to overpressure, before starting any sequence.

Month ##, 200X


Agilent Restricted

Mobile Phase and Sample Recommendations to

Avoid High Pressure

If the system has been sitting with buffer in it, flush the injector as well as the

column. This prevents any bacterial growth in the injector from transferring to

the column.

Replace bottles of mobile phase buffer every 24 – 48 hours. Do not top off the

bottle with more mobile phase, replacing the buffer with a fresh bottle

Do not use a high buffer salt mobile phase (>50mM) in combination with high

ACN concentrations due to possible precipitation.

Filter all aqueous buffers prior to use through a 0.2 um filter.

Use solvents that are high quality chromatography grade solvents (HPLC or

MS grade).

Filter all samples with particulates through an appropriate 0.2um filter.

Particulates can clog the inlet frit on the column and cause high pressure and

short column lifetime.

Month ##, 200X


Agilent Restricted

How to Measure Dwell Volume and

Extra Column Volume

Dwell Volume

Gradient Separations What is Dwell Volume?

• Dwell volume = volume from formation of gradient to the column • Behaves as isocratic hold at the beginning of gradient.

{

0 10 20 30 40

Minor Dwell Volume Differences

Can Change Resolution

0 10 20 30 40

VD = 0.43

mL

Column: ZORBAX Rapid Resolution Eclipse

XDB-C8

4.6 x 75 mm, 3.5 µm

Mobile Phase: Gradient, 0 - 100 %B in 52.5

min.

A: 5/95 methanol/ 25 mM phosphate

pH 2.50

B: 80/20 methanol/25 mM phosphate

pH 2.50

Flow Rate: 0.5 mL/min

Temperature: 25°C

Injection: 5 L

Detection: 250 nm

Sample: Mixture of antibiotics and antidepressants

Upper trace simulates actual run data

entered into DryLab® 3.0 software

Lower trace is simulated chromatogram for

larger VD

VD = 2.0

mL

001814S1.PPT

Determining the Dwell Volume

of Your System

Replace column with short piece of HPLC stainless steel tubing

Prepare mobile phase components

A. water - UV-transparent

B. water with 0.2% acetone - UV-absorbing

Monitor at 265 nm

Adjust attenuation such that both 100% A and 100% B are on scale

Run gradient profile 0 - 100% B/10 min at 1.0 mL/min

Record

Best straight-line fit

through linear trace

Extension of

original baseline

Intersection indentifies

dwell time (tD)

(tD)

0 10 20

Re

sp

on

se

VD = tD x F

VD = Dwell Volume

Time (min)

001815S1.PPT

Measuring Dwell Volume

High Pressure Mixing: VD = mixing chamber + connecting tubing + injector

Low Pressure Mixing: VD = the above + pump heads + associated plumbing

Re

sp

on

se

0 10 20

Best straight-line

fit through linear

trace

Extension of

origional baseline

tD Time (min)

Intersection

identifies dwell

time (tD)

VD = tD x F

tD : Imposed Isocratic Hold Typical VD = 0.5 - 15 mL

If using gradient conditions - report dwell volume (VD)

VD varies from instrument to instrument

001011P1.PPT

Dwell

Volume

Impact A chromatogram

generated on one

instrument (VD1) can have

a very different profile if

generated on another

instrument (VD2)

Measuring Dwell Volume

1. Measure the Dwell Volume of your HPLC System

VD = 1.0 mL

2. Draw Effective Gradient Profile at First Flow Rate

Calculate the time delay (imposed isocratic hold)

caused by dwell volume

VD = tD F 1.0 mL = tD 1.0 mL / min

where F = 1.0 mL / min for 4.6 x 150 mm column

VD = 1.0 mL

tD = F/VD tD = 1.0 mL / min / 1.0 mL

tD = 1.0 min

Injection

Start of Gradient t = 1.0min

0 - 60%

000884P2.PPT

Correcting for Dwell Volume

3. Draw Effective Gradient Profile at Second Flow Rate

tD = F / VD tD = (0.2 mL / min) / 1.0mL

tD = 5.0 min

where F = 0.2 mL / min for 2.1 x 150 mm column

VD = 1.0 mL (same for HPLC system)

Injection

Start of Gradient t = 5.0min

t = 0

Delayed

Injection t = 4.0 min

Delay injection on the 2.1 x 150 mm column

by 4.0 min (5.0 min - 1.0 min) so that the gradient

profile is the same on both columns

001013P1.PPT

To Accommodate Different Column Sizes

If VD1 > VD2 Compensate for longer VD1 by adding

an isocratic hold to VD2, such that

Hold + VD2 = VD1

If VD1 < VD2

Delay injection, such that VD2 - delay = VD1

( very difficult to accomplish in practice )

001014P1.PPT

Correcting for Dwell Volume

Gradient Separations What is Extra-Column Volume?

• Extra Column Volume = volume from Injector+Connecting Tubing+Detector Cell Volume

• All volume outside of the column allows dispersion with resulting band broadening and loss of resolution.

Remove HPLC column from instrument

Join injector and detector tubing with

zero-dead-volume union

Inject (0.5 - 2 L) of toluene in 100% acetonitrile

Determine width of peak at base (winstrument)

Peak bandwidth follows:

One Way:

w2tot = w2

col + w2instrument

001812S1.PPT

How to Estimate the Extra Column Volume of an

HPLC System

Instrument #2

winstrument = 83 L

Instrument #1

Low Volume System

winstrument = 42 L

Toluene in Acetonitrile

Time (min)

0 0.08 0.16 0.24 0.32 0.40 0.48

For peak having a k = 2

4.6 x 150 mm, 5 m w2

tot = (180)2 + (42)2 w2tot = (180)2 + (83)2

wtot = 185 L wtot = 198 L

4.6 x 50 mm, 3.5 m w2

tot = (73)2 + (42)2 w2tot

= (73)2 + (83)2

wtot = 84 L wtot = 110 L

Time (min)

0 0.08 0.16 0.24 0.32 0.40 0.48

w2tot = w2

col + w2instrument

3%

15%

51%

10%

001813S1.PPT

How to Estimate the Extra Column Volume of an

HPLC System

Delayed Injection is done by an Injector Program

implementation of high resolution fast lc · pdf file · 2015-07-28power n(5...

Documents