Implementation of High Resolution Fast LC - Agilent
Transcript of 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
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Analysis Time*
-33%
-50%
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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
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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
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• 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
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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
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4.6 x 100 mm, 3.5 µm
1
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Rs3,2= 6.65
Rs3,2= 6.414.6 x 150 mm, 5 µmP=82 bar
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020406080
100
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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
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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 =��
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ml/minmmmm
ml/min 21.0 2.30 1.05
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Reducing Column Size? Reduce Injection Volume!
2 col.column1
column21 col. Inj.Vol.
VolumeVolume
Inj.Vol. =��
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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 =��
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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
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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 mm 50 mm = 0.5 x Original
150 mm 1100 mm = 0.67 x Original
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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
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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
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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
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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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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
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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
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Group/Presentation TitleAgilent Restricted
Month ##, 200XPage 22
� � �0 2 4 6 8 10 12 14 16 180
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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
0.17 x 400 mm capillary
0.17 x 150 mm capillary
Thermostatted
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0.12 x 150 mm capillary
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
0.17 x 105 mm capillary
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
0.12 x 105 mm capillary
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
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System Tubing Volume Not Optimized0.17mm i.d. tubing
Peak width 0.038 min
Peak width 0.037 min
Resolution 0.961
min0.5 1 1.5 2 2.5
mAU
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550
600 System Tubing Volume Optimized0.12mm i.d. tubing
Peak width 0.018 min
Peak width 0.019 min
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
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Group/Presentation TitleAgilent Restricted
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
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0
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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%
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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
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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
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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
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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
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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
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1 501 1001 1501 2001 2501
Injections
Pre
ssur
e
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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
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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
Group/Presentation TitleAgilent Restricted
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
Group/Presentation TitleAgilent Restricted
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
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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
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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