Implementation of High Resolution Fast LC - Agilent

Implementation of High Resolution Fast LC

Great idea but what will I have to change?

Agilent Technologies 2012High 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

Running Faster –What Instrument Settings Are Important?

Isocratic Speed

• Flow Rate• Pressure• Temperature• Detection

Gradient Speed

• Flow Rate and Gradient Time• Pressure• Temperature• Detection• Dwell Volume (Re-Equilibration Time)

What Is Your Current Column Efficiency and How Fast Do You Want To Run?

ColumnLength(mm)

Resolving Power

N(5 µm)

Resolving Power

N(3.5 µm)

ResolvingPower

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

��

Analysis Time*

-33%

-50%

Page 4

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

��

-50%

-67%

-80%

-90%��

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�� !"

“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

Steps for Increasing Isocratic HPLC Speed

Step 1 (Easy)

• Reduce Column Length and Particle Size

• Maintain Flow Rate

Step 2 (Easy)

• If Pressure is Within Limit, Increase Flow Rate

• Approaching Pressure Limit of Instrument ? Increase Temperature to Lower Pressure Temperature to Lower Pressure

• Increase Flow Rate

Step 3 (Moderate Instrument Alterations)

• Approaching Flow Rate Limit of Instrument ?

• Decrease Column Diameter and Reduce Flow Rate Proportionately

• Need to Reduce Injection Volume Based on Ratio of Column Volume

ISOCRATIC ELUTION

Page 6

• When flow limit of pump is reached

• When approaching about 80 - 90% pressure limit of instrument

When to Stop!?

• When resolution is no longer satisfactory

Reduce Column Length/Particle Size by Same Ratio

5um 1.8um

Reduce column length by factor of 3

Page 8

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: 6uLTemperature: 25�C Flow: 1 mL/min. Detection: 310, 4 nm, 0.5 s response time, semi-micro flow cell, Sample: Sunscreens

� ��0 2 4 6 8 10 12 14

� ��

0

20

40

60

� ��

4.6 x 100 mm, 3.5 µm

1

2 3

4

Rs3,2= 6.65

Rs3,2= 6.414.6 x 150 mm, 5 µmP=82 bar

� ��0 2 4 6 8 10 12 14

020406080

100

� ��0 2 4 6 8 10 12 14

� ��

0

50

100

150

4.6 x 100 mm, 3.5 µmP=105 bar

Rs3,2= 6.514.6 x 50 mm, 1.8 µmP=208 bar

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

4.6 mm 3 mm

Reduce flow rate by factor of 0.4

Page 10

2.1 mm4.6 mm

Reduce flow rate by factor of 0.2

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

2 col.

2

column1

column21 col. Flow

RadiusRadius

Flow =��

�

��

�×

ml/minmmmm

ml/min 21.0 2.30 1.05

1.0 i.e.2

=��

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�×

Page 11

Reducing Column Size? Reduce Injection Volume!

2 col.column1

column21 col. Inj.Vol.

VolumeVolume

Inj.Vol. =��

�

��

�×

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

2 col.column1

column21 col. 4

2.04.0

20 i.e. �lmlml

�l =��

�

��

�×

Page 12

Zorbax column volume = 3.14 x r 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 mm4.6 mm

= 0.4 x Original

= 0.2 x Original

xReduction to allow for length change

Page 13

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



��

100% B

100% B

100% B

tg= 10

tg= 5

tg F

S ∆Φ ∆Φ ∆Φ ∆Φ Vmk* ∝

0% B

0% B

0 10 20 30 40

Time (min)

100% B

tg= 40

tg= 20 F = flow rate (mL/min.)tg = gradient time (min.)

Vm = column volume (mL)

∆Φ ∆Φ ∆Φ ∆Φ = % B change S = constant

0% B

0% B

Simplified Method Transfer for Increased Gradient Speed

Step 1

• Reduce Column Length and Particle Size

• Adjust Gradient Time by same Factor

• Maintain Flow Rate

Step 2

• If Pressure is within Limits, Increase Flow and Reduce Gradient Time

GRADIENT ELUTION

• Stop When Reach Flow Limits of Instrument

Step 3

• Decrease Diameter of Column

• Match Flow to New Column Diameter

• Reduce Injection Volume

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

Page 15

Example of Possible Speed Increase

F= 1.20ml/minT = 40�C

Analysis Time = 11min 4.6mm x 150mm 5.0µm

min0 2 4 6 8 10 12

F = 4.80ml/minT = 40�C

Analysis Time = 1.05min 4.6mm x 50mm 5.0µm

min0 0.2 0.4 0.6 0.8 1

Page 16

min0 0.2 0.4 0.6 0.8 1

F= 1.00ml/minT = 40�C

Analysis Time = 1.1min2.1mm x 50mm 1.8µm

min0.2 0.4 0.6 0.8 10

Max Speed at T = 95oC2.1mm x 50mm 1.8um

F= 2.40ml/minT = 95�C

Analysis Time: 0.4minPWHH = 197msec

min0.2 0.4 0.6 0.8 10

> 20x faster !

Optimizing Gradient Separations with 1.8 um RRHT Columns: 10 X Faster Analysis

min0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25

RRHT SB-C182.1 x 50mm, 1.8um

Temp: 50��CFlow: 1 mL/min

Gradient (tG): 2.4 min

Rapid Resolution SB-C183.0 x 150mm, 3.5um

Page 17

min5 10 15 20 25

3.0 x 150mm, 3.5umTemp: 25��C

Flow: 1.4 mL/minGradient (tG) : 18 min

SB-C184.6 x 250mm, 5um

Temp: 25��CFlow: 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

150mm100mm

• Broken lines are isobar (800 bar)50mm

Shorter Columns with Fast GradientsYield Higher Peak Capacity

50mm

150mm100mm

Shorter Gradient (5 min)

Peak Capacity:• 258 for 50 mm• 240 for 100 mm• 221 for 150 mm50mm • 221 for 150 mm

Separation of 12 Phenols on Poroshell 120 EC-C18 5 minutes – 50mm Column

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

Mobile Phase:Solvent A: Water with 0.1% Formic Acid

Solvent B: AcetonitrileGradient::

Time %B0.8 5%

6.8 60%1200 SL controlled temperature at 25 C

2 mm flow cell

1. Hydroquinone2. Resourcinol3. Catechol4. Phenol5. 4-Nitrophenol6. p-cresol7. o-cresol8. 2-Nitrophenol9. 3,4 di methyl phenol10. 2,3 di methyl phenol11. 2,5 di methyl phenol12. 1-napthol

274 bar 2.5 ml/min

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0 5

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

150mm100mm

• 525 for 150 mm50mm

Constant Particle Size, Gradient TimeMore Resolution with Longer Column

� � �0 2 4 6 8 10 12 14 16 18

�

0

50

100

150

200

�

200

RRHD SB-C18 2.1 x 50 mm, 1.8umPmax=366 barnc = 424

RRHD SB-C18 2.1 x 100 mm , 1.8umRs: 1.37

Rs: 0

Page 22

Group/Presentation TitleAgilent Restricted

Month ##, 200XPage 22

� � �0 2 4 6 8 10 12 14 16 180

50

100

150

� � �0 2 4 6 8 10 12 14 16 18

�

0

50

100

150

200

RRHD SB-C18 2.1 x 100 mm , 1.8umPmax=595 bar nc = 485

RRHD SB-C18 2.1 x 150 mm, 1.8umPmax=768 bar nc = 589

Rs: 2.40

Rs: 1.37

HPLC Instrument Components

Gradient Delay or Dwell Volume.

Extracolumn Volume

Data Sampling or Acquistion Rate

.

Number of Scans Number of Scans or pointsor points

Minor Dwell Volume DifferencesCan Change Resolution

VD = 0.43 mL

Column: ZORBAX Rapid Resolution Eclipse XDB-C84.6 x 75 mm, 3.5 µm

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

A: 5/95 methanol/ 25 mM phosphatepH 2.50 B: 80/20 methanol/25 mM phosphatepH 2.50

Flow Rate: 0.5 mL/min

#�$��%�

0 10 20 30 40

0 10 20 30 40 Temperature: 25�CInjection: 5 µL

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

High pressureGradient pump

Std or WellPlate sampler

Standard assembly without standard mixer

0.12 x 400 mm capillary

High pressure Gradient pump

Std or WellPlate sampler



Thermostatted

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µ Thermostatted

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Diode Array Detector

DAD equipped with a 1.7 µL flow cell

Mass Spectrometer

0.12 x XX mm PEEK Capillary

Rapid ResolutionHT Column

Diode Arraydetector

Waste


DAD equipped with a 5uL or 1.7 µL flow cell

3 µL heat exchanger

Thermostatted Column

compartment

Cell Inlet Capillary

Cell Outlet Capillary

Rapid ResolutionHT Column


3 µL heat exchanger

Thermostatted Column

compartment

Cell Inlet Capillary

Cell Outlet Capillary

Why Optimize System Volume?

min0.5 1 1.5 2 2.5

mAU

0

100

200

300

400

System Tubing Volume Not Optimized0.17mm i.d. tubing

Peak width 0.038 min


Resolution 0.961

min0.5 1 1.5 2 2.5

mAU

0

100

200

350

400

550

600 System Tubing Volume Optimized0.12mm i.d. tubing



Resolution 1.902

min0.5 1 1.5 2 2.5

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

F = 2 ml/minP = 975 bar

0.5min

� �

0

100

200

300

400

500


Month ##, 200XPage 27 June, 2011June 06, 2005Page 27

F = 2.3 ml/minP = 1110 bar

0.4min

� ��

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.450

� ��0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

� �

0

100

200

300

400

500

300 Pesticides < 20 minutes, 1290 Infinity

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

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

Ultrafast LC/MS Analysis for 15 Analyte Subset

Peak Width 0.7 sec

RRHD Eclipse Plus C182.1x 50 mm, 1.8 um

750 bar1 minute

Time Composition

1290 Infinity Applications

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

Volume of the Column:

• V=�(2.1/2)2(50)=173.2 µL

Optimized 1290

Total Extra Column Volume:

• 1.1+1.1+0.8=3 µL

Volume of the Column:

• V=�(2.1/2)2(50)=173.2 µLVoid Volume of the Column:

• 173.2*0.6=103.9 µL

Percent Extra-Column Volume:

• (8.6/103.9)100=8.3%

• V=�(2.1/2) (50)=173.2 µL

Void Volume of the Column:

• 173.2*0.6=103.9 µL

Percent Extra-Column Volume:

• (3/103.9)100=2.9%

Page 30

Effect of Extra-column Volume on a Gradient Analysis of Alkylphenones

Default 1290, 8.6 µL Extra-column Volume Pmax=320 barRs5,6=1.18

nC=35

Page 31

Optimized 1290, 3.0 µL Extra-column Volume

Pmax=323 barRs5,6=2.25 +91%

nC=56+60%

What Happens If the Connections Poorly Made ?

Ferrule cannot seat properly

Wrong … too long

Wrong … too short

Page 32

If Dimension X is too long, leaks will occur

Mixing Chamber

If Dimension X is too short, a dead-volume, or mixing chamber, will occur

X

X

min0 0.1 0.2 0.3 0.4

mAU

020406080

100120140 One bad capillary connection!130 mAU

Influence of Bad Post-Column Connection

min0 0.1 0.2 0.3 0.4

mAU

0306090

120150180210 Fixed!

160 mAU

Page 33

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

80Hz

PW=0.30sec

40H

PW = 0.33 sec

80Hz vvsvs.us 20Hz– 30% Peak Width+30% Resolution

+ 40% Peak Capacity+ 70% Apparent Column

min0.1 0.2 0.3 0.4 0.50

40Hz

20 Hz

PW=0.42sec

10Hz

PW=0.67sec

5Hz

PW=1.24sec

•

+ 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

Diflusinal in Plasma

320

340

360

380

400

160000

180000

200000

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 minutesNot Centrifuged/ Not Filtered

Injection Volume: 1ul injections

Solvent A: Water w/0.1 % TFASolvent B: MeCN w/0.08 % TFA

Flow Rate 1 ml/min 1 ul injectionTime % B0 200.5 900.6 90

35

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

1 501 1001 1501 2001 2501

Injections

Pre

ssur

e

0

20000

40000

60000

80000

100000

120000

140000

Eff

icie

ncy

(N)

End PressPlates

0.6 901.1 202.5 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 valvesmoving parts of injection valves

• Results in less downtime of the instrument for repairs

• Produces improved analytical results by removing potentially interfering contamination

Page 36

In-Line Filters Provide Good Insurance Against System OverPressure

Page 37

Summary

APPENDIX

RRLC – A Tip for Controlling Unnecessary PressureA 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 FiltersMore Necessary Than With 3.5u and 5.0u Particle Columns

Page 40

RRHT Column Installation Recommendations to Avoid Complaints of High Pressure1. 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.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


Mobile Phase and Sample Recommendations to Avoid High PressureIf 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 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


Low and High Pressure MixingLow and High Pressure Mixing

• Point of mobile phase mixing

before pump head

July 12, 2012Page 43

• Point of mobile phase mixing after

pump head• 120 ul without damper or mixer

• 320 ul with 200ul

Agilent 1260 Infinity Quaternary and Binary PumpsAgilent 1260 Infinity Quaternary and Binary Pumps

Low-pressure mixing (LPM) by proportioning valve before the pump head

Quaternary PumpQuaternary Pump

High-pressure mixing (HPM) Combination and mixing of

mobile phases after the pump heads

Binary PumpBinary Pump

System System –– Signal height Signal height System volumes System volumes –– Delay volumeDelay volume

Delay volume ~ 700 �L

min0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

100

200

300

400

120 mAU

min0.5 1 1.5 2 2.5 3 3.5 4 4.5

Delay volume ~ 120 �L

min0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

100

200

300

400

Column: ZORBAX SB-C18 2.1 x 50 mm, 1.8 �mFlow: 0.42 mL/min

220 mAU

Effects of Delay Injection ProgramEffects of Delay Injection Program

Delayed Injection is done by an Injector ProgramDelayed Injection is done by an Injector Program

Documents

Implementation of High Resolution Fast LC - Agilent