resolution: too much, too little, or just right? · 2019. 1. 17. · resolution & the...
TRANSCRIPT
![Page 1: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/1.jpg)
Resolution:
Jean LaneApplications Engineer:
LC Columns and Consumables Technical Support
January 15, 2019
January 14, 2019
Too Much, Too Little or Just Right
1 For Research Use Only. Not for use in diagnostic procedures.
![Page 2: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/2.jpg)
Topics for Discussion:
Resolution & the Resolution Equation
• Parameters defining resolution
• Importance of these parameters
Getting the Desired Resolution
• Know your requirements
• Changing individual parameters to effect resolution
Mobile Phase & Gradient
• Considerations for mobile phase, i.e. pH
Choice of organic
• Gradient elution and the resolution relationship
Role of the Instrument
• Delay volume
• Dispersion
• Column Temperature
• Detector Flow Cell Volume
• Data Collection Rate and Importance
For Research Use Only. Not for use in diagnostic procedures.January 14, 20192
![Page 3: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/3.jpg)
Resolution Definition
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.3
Resolution is a measure of the ability to separate two
components
![Page 4: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/4.jpg)
Basic Chromatography Parameters
Equations Describing Factors Controlling RS
k =(tR-t0)
t0
α = k2/k1
N = 16(tR / tW)2
Theoretical Plates-Efficiency
Selectivity or Separation Factor
Retention Factor
Feb 19, 2014 For Research Use Only. Not for use in diagnostic procedures.4
![Page 5: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/5.jpg)
N = Column Efficiency – Column length and particle size
a = Selectivity – Mobile phase and stationary phase
k = Retention Factor – Mobile phase strength
Resolution …
Determined by 3 Key Parameters –
Efficiency, Selectivity and Retention
The Fundamental Resolution Equation
w
∆tR=
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.
Resolution Equation
5
![Page 6: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/6.jpg)
Factors that Improve Resolution
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.6
![Page 7: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/7.jpg)
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.7
Retention Factor (k):
Resolution (Rs) & Parameters Affecting Resolution
Describes how well an analyte is retained by the stationary phase.
This is expressed as a ratio of column volumes.
This can be adjusted by making changes to the organic strength of the mobile phase
![Page 8: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/8.jpg)
Resolution (Rs) & Parameters Affecting Resolution
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.8
Column Efficiency as Theoretical Plates (N)
As the number of plates increase, peaks become thinner and sharper, which
improves resolution.
Plates are often described by their height (H), or Height Equivalent to the
Theoretical Plate (HETP)
Number of plates and plate height are inversely proportional, i.e. H = L/N
![Page 9: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/9.jpg)
Resolution (Rs) & Parameters Affecting Resolution
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.9
Selectivity or Separation Factor (α)
• This is the ratio of retention factors for two adjacent peaks.
• Larger α values indicate better separation.
• Selectivity can be adjusted by changes to either the mobile
phase or the stationary phase.
![Page 10: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/10.jpg)
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.10
Factors That Effect Resolution
![Page 11: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/11.jpg)
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.11
Getting the Desired Resolution:Resolution is a Key Goal in Chromatographic Separations
![Page 12: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/12.jpg)
How Much Resolution is Necessary?
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.12
• Insufficient Rs will compromise accuracy, precision, robustness, and ruggedness
• Initial resolution can decrease due to changes in separation variables
• Build in robustness so that ΔRs is small when separation variables are changed
![Page 13: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/13.jpg)
Increase Resolution with no run time increase
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.13
1. For smaller particles the height of the theoretical plate decreases.
2. Therefore the number of theoretical plates in a column with a given
length increases.
3. In addition, note optimal flow rate for smaller particles is increased.
![Page 14: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/14.jpg)
Reduce Particle Size & Maintain column length
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.14
Increased ‘N” in an isocratic separation – improved resolution
![Page 15: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/15.jpg)
Maintaining Resolution
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.15
Reduce column length AND Particle size
![Page 16: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/16.jpg)
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.16
Selectivity and Column ChoiceEvaluate Different Bonded Phases
![Page 17: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/17.jpg)
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.17
Differences in SelectivityAgilent InfinityLab Poroshell Bonded Phases
![Page 18: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/18.jpg)
Importance of Alternate Selectivity Chemistries
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.18
Agilent InfinityLab Poroshell 120 columns 4.5 x 40 mm, 2.7µm
70/30 – MeOH/H2O, 1.5mL/min, 40C, 254nm
![Page 19: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/19.jpg)
Polar Embedded Phase for Alternate Selectivity:
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.19
Agilent InfinityLab Poroshell 120 EC-C18
Agilent InfinityLab Poroshell 120 Bonus RP
10-70 % methanol/12 min, DAD 260 nm, 0.35 mL/min, 2.1 x 100 mm, 40°C, 10 mM, pH 3.8 ammonium formate buffer and methanol
![Page 20: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/20.jpg)
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.20
Agilent InfinityLab Poroshell 120 Portfolio
Best all around
Best for low
pH mobile
phases
Best for high
pH mobile
phases
Best for
alternative
selectivity
Best for polar
AnalytesBest for Chiral
InfinityLab Poroshell
120
EC-C18
1.9 µm, 2.7 µm, 4
µm
InfinityLab Poroshell
120
SB-C18
2.7 µm
InfinityLab Poroshell
HPH-C18
1.9 µm, 2.7 µm, 4
µm
InfinityLab Poroshell
120 Bonus-RP
2.7 µm
InfinityLab Poroshell
120
HILIC
1.9 µm, 2.7 µm, 4
µm
InfinityLab Poroshell
120
Chiral-V
2.7 µm
InfinityLab Poroshell
120
EC-C8
1.9 µm, 2.7 µm, 4
µm
InfinityLab Poroshell
120
SB-C8
2.7 µm
InfinityLab Poroshell
HPH-C8
2.7 µm, 4 µm
InfinityLab Poroshell
120
PFP
1.9 µm, 2.7 µm, 4
µm
InfinityLab Poroshell
120
HILIC-Z
2.7 µm
InfinityLab Poroshell
120
Chiral-T
2.7 µm
InfinityLab Poroshell
120 Phenyl-Hexyl
1.9 µm, 2.7 µm, 4
µm
InfinityLab Poroshell
120
HILIC-OH5
2.7 µm
InfinityLab Poroshell
120
Chiral-CD
2.7 µm
InfinityLab Poroshell
120
SB-Aq
2.7 µm
InfinityLab Poroshell
120
Chiral-CF
2.7 µm
InfinityLab Poroshell
120
EC-CN
2.7 µm
Choice of 18 chemistries
![Page 21: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/21.jpg)
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.21
When does pH Affect Selectivity and Resolution?
Example of Compound Type Comparison
Nonpolar IonizablePolar
If an ionizable compound, acids and bases can change retention and selectivity with changes in pH
![Page 22: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/22.jpg)
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.22
Agilent InfinityLab Poroshell Column
HPH C18, 2.7um
Change in Retention with pH for Ionizable Compounds
Is Compound Dependent
![Page 23: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/23.jpg)
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.23
Selectivity can be controlled by Changing pH
Agilent InfinityLab Poroshell HPH C18 4.6 x 50mm, 2.7µm
![Page 24: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/24.jpg)
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.24
Change in Retention w/pH for Ionizable Compounds
is Key to Method Development
![Page 25: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/25.jpg)
Mobile Phase & GradientsEvaluate Organic Modifiers
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.25
MeOH – higher pressure, generally better peak
shape with bases, protic solvent
Acetonitrile – lower pressure, wider UV window,
stronger than MeOH
WHY?
✓ It’s easy – ACN & MeOH are readily
available
✓ Works on any bonded phase – optimize
separation no matter the column choice
![Page 26: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/26.jpg)
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.26
Further Optimization of Organic to Improve Resolution
Method tested for reproducibility for resolution
Slight Change to Method can give more reproducible results
![Page 27: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/27.jpg)
Resolution Relationship for Gradient Elution
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.27
![Page 28: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/28.jpg)
This Relationship says that to Keep Relative Peak Positionin the Chromatogram unchanged..
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.28
![Page 29: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/29.jpg)
Conventional Column – 4.6 x 150mm, 5µm SB-C18
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.29
![Page 30: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/30.jpg)
Maintaining Peak Position & Resolution
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.30
Have shortened column & Gradient time – Need to do so by the SAME factor
1/3 column length – 1/3 Gradient time
ex: RRHT column – 4.6 x 50 mm, 1.8µm, SB-C18
![Page 31: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/31.jpg)
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.31
Gradient Steepness Effects Resolution & Retention (k*)
![Page 32: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/32.jpg)
Gradient Steepness and Gradient Shape
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.32
1. Gradient shape
• Linear gradients are preferred
• Nonlinear, segmented, and step gradients can be used but can be harder to transfer
2. Gradient Steepness
• Change can affect resolution
• Compare resolution at desired gradient time and at tg +/- 10-20%
• Small changes likely due to instrument performance differences
• Need to compensate for any dwell/delay volume differences first_
![Page 33: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/33.jpg)
Instrument: Areas of Instrument that Impact ResolutionVolume Considerations for LC systems
For Research Use Only. Not for use in diagnostic procedures.33 January 14, 2019
1. Delay volume
2. Dispersion volume/Extra Column Volume
3. Temperature
4. Detector Flow cell volume
5. Data collection rate Temperature
Detector
Flow Cell
![Page 34: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/34.jpg)
Gradient Delay VolumeSystem Design – Agilent 1290 Infinity II LC System
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.34
Affects our results:
• An isocratic hold step at the beginning of every gradient
• Sharpness of the gradient
• Required equilibration time and therefore total cycle time
Early eluting peaks are more affected than later eluting peaks
![Page 35: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/35.jpg)
Gradient Delay VolumeAgilent 1290 vs 1200 systems
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.35
Mixing Behavior
![Page 36: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/36.jpg)
Instrument Delay Volume DifferencesCan Cause Changes in Resolution & Retention
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.36
2.1 x 100 mm Agilent ZORBAX Eclipse Plus, 1.8 µm, flow = 0.8 mL/min
![Page 37: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/37.jpg)
Instrument ConsiderationsDispersion & how it can effect resolution
What is dispersion?
• Original sample concentration being diluted as it is carried through the system plumbing (extra-column volume)
What increases dispersion?
• Connecting tubing that is too long
• Connecting tubing that is too large in diameter
• Connections that have gaps and form small mixing chambers
January 14, 201937 For Research Use Only. Not for use in diagnostic procedures.
![Page 38: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/38.jpg)
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.38
ECV is the volume in the LC system outside of the column
@ Column Compartment
@ Autosampler@ Detector
Dispersion & ECV : where on LC might it be?
![Page 39: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/39.jpg)
Optimizing Connecting Tubing Volume For UHPLC Columns
January 14, 201939
min0.5 1 1.5 2 2.5
mAU
0
100
200
350
400
550
600 System Tubing Volume Optimized
0.12mm id tubing
Peak width 0.018 min
Peak width 0.019 min
Resolution 1.902
min0.5 1 1.5 2 2.5
mAU
0
100
200
300
400
System Tubing Volume Not Optimized
0.17mm id tubing
Peak width 0.038 min
Peak width 0.037 min
Resolution 0.961
Length 10mm 50mm 100mm 150mm
Tubing ID Volume Volume Volume Volume
0.17mm
(green)
0.227 uL 1.1uL 2.27 uL 3.3 uL
0.12mm (red) 0.113 uL 0.55uL 1.13 uL 1.65 uL
For Research Use Only. Not for use in diagnostic procedures.
![Page 40: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/40.jpg)
40 January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.
Resolution & Peak ShapeEffect of Extra Column Volume
2.1 x 150 mm columns
Flow rate: 0.2mL/min
Extra-
Column
Volume
Improved
Resolution
![Page 41: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/41.jpg)
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.41
Extra Column Volume – Resolution & Peak Shape Lost
Resolution lost
Resolution
improved
![Page 42: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/42.jpg)
Making Correct Connections
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.42
Poor Fitting Connections
• Will broaden or split peaks or cause tailing
• Will typically affect all peaks, but especially
early eluting peaks
• Can cause carry-over
![Page 43: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/43.jpg)
Importance of Correct Connections
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.43
Agilent Technical Note: Agilent InfinityLab UHPLC Fittings
Pub No 5991-5525EN
Correct connection every time
Quick Connect
Quick Turn Compatible to 1300 bar
![Page 44: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/44.jpg)
Evaluating Temperature
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.44
Column Temperature
Adequate Temperature Control is Essential
![Page 45: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/45.jpg)
Temperature Can Optimize Resolution and Selectivity
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.45
Gradient of Ten Cardiac Drugs on Zorbax SB C18 RRHT
![Page 46: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/46.jpg)
Temperature Can Also be used to Optimize a HILIC Separation
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.46
![Page 47: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/47.jpg)
Scenario: Agilent ZORBAX Rapid Resolution Column: 75 mm, 3.5 µm; Flow Rate: 1mL/min; k = 3
1.7 µL
8 µL
14 µL
0.3%
6%
19%
Flow Cell
Volume
Band Broadening* (4.6 mm)
6%
138%
423%
Band Broadening* (2.1 mm**)
*Versus 8571 theoretical plates (HPLC Calculations Assistant, Version 2.1, Savant Audiovisuals)
**Flow Rate, 0.2 mL/min
Differences in Detector Flow Cell Volume Can Affect N and Rs
Resolution & Importance of Flow Cell Volume
January 14, 201947 For Research Use Only. Not for use in diagnostic procedures.
![Page 48: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/48.jpg)
48 January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.
System Data Collection RateOptimize for peak Rs
![Page 49: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/49.jpg)
Maintaining Resolution at High Analysis Speed
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.49
Importance of data collection rate for narrow peaks
![Page 50: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/50.jpg)
In Summary:
January 14, 2019 For Research Use Only. Not for use in diagnostic procedures.50
Resolution Equation• Become familiar with the Resolution equation and its parameters
Efficiency, retention, and selectivity. Selectivity is the main driver
of resolution.
Required Resolution• Baseline resolution is achieved at 1.5 but need to strive to achieve resolution of > 2.0.
Consider Alternates for Improving Resolution• Explore alternate selectivity by choosing and evaluating different bonded phases.
• Also look to selectivity effects of both mobile phase organics and pH.
Gradient time & Steepness• Optimal gradient is one where we have sufficient (i.e. >2.0) resolution at the shortest runtime.
Role of the Instrument• From System delay volume, ECV & Dispersion, to correct fittings, temperature, flow cell &
data collection rate, the LC system too needs to be optimized to ensure that acceptable resolution
is achieved and maintained.
![Page 51: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/51.jpg)
THANK YOU FOR ATTENDING
ANY QUESTIONS??
For Research Use Only. Not for use in diagnostic procedures.January 14, 2019
515151
51
![Page 52: Resolution: Too Much, Too Little, or Just Right? · 2019. 1. 17. · Resolution & the Resolution Equation • Parameters defining resolution • Importance of these parameters Getting](https://reader034.vdocuments.mx/reader034/viewer/2022051907/5ffa8915e651472a9e464742/html5/thumbnails/52.jpg)
For Research Use Only. Not for use in diagnostic procedures.
Contact Agilent Chemistries & Supplies Technical Support
• 1-800-227-9770 Option 3, Option 3:• Option 1 for GC/GCMS Columns and Supplies
• Option 2 for LC/LCMS Columns and Supplies
• Option 3 for Sample Preparation, Filtration, and QuEChERS
• Option 4 for Spectroscopy Supplies
*available 8am – 5pm EST – PST in US and Canada
January 14, 201952