Tips and Tricks of Faster LC Analysis Without Capital InvestmentCapital InvestmentIt may be easier than you think!
A il t T h l i IAgilent Technologies, Inc.
July 14, 2009
Page 1
There Are Many Reasons to Reduce Analysis Time
• Run More Samples/Day
• Reduce Mobile Phase Use• Reduce Mobile Phase Use
• Reduce Mobile Phase Waste Disposal
• Better Utilize Existing Resources in Instruments and Personnel• Better Utilize Existing Resources in Instruments and Personnel
• Implement “HB5”* Work Schedule
(* “HB5” H B 5(* “HB5” Home By 5, Ray Lombardi, Agilent Technologies, Inc. 2007)
At what cost and difficulty?At what cost and difficulty?That depends on what you want to accomplish.
Page 2
How Fast Do You Want To Go?Your Answer Will Determine the Cost of Increased SpeedYour Answer Will Determine the Cost of Increased Speed
• 2 to 3 X increase in Speed – Easy
• 5-10 X Increase – May require instrument tweak
• 10 X + Increase – Will require instrument upgrade and tweak
What are the paths to LC method speed gains?
• Most speed from column and particle size optimization
• Instrument optimization will enable or boost those gains
The following discussion is designed to simplify the process; Often at no cost!
Page 3
How Do Small Particles Help Speed Up Methods?Help Speed Up Methods?
• Increase efficiency (N) in same length columny ( ) g
• Allow shorter column to match efficiency of longer ..column
• Provide expanded flow rate range without loss of ..efficiency
Page 4
Trick: Use Smaller Particles For More Efficient Separations Across a Wide Flow Rate RangeSmall Particle Columns including Monolith
0.0030Dimensions: 4.6 x 50/30/20mmEluent: 85:15 ACN:Water
/
ZORBAX 5.0μmZORBAX 3 5μm
0.0020
0.0025 Flow Rates: 0.05 – 5.0 mL/minTemp: 20°CSample: 1.0μL Octanophenone in Eluent
ate)
ZORBAX 3.5μmVendor A 2.5μmVendor B 2.0μmZORBAX 1.8μmMonolith
0.0015
TP (c
m/p
la Monolith
0 0005
0.0010HET
0.0000
0.0005
0 0 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0
Page 5
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0Volumetric Flow Rate (mL/min)
ISOCRATIC ISOCRATIC ELUTION
Page 6
ResolutionResolution
N k 1⎛⎜ ⎞⎟−⎛⎜ ⎞⎟
αR N kks =
4 11
+⎛⎝⎜
⎞⎠⎟⎛⎝⎜
⎞⎠⎟
ααk4 1+⎝ ⎠⎝ ⎠α
Tip: Short Column/Smaller Particle Maintains RsTrick: Tailor Column to Analysis Needs
ColumnLength
Resolving Power
N(5 )
Resolving Power
N(3 5 )
ResolvingPower
N(1 8 )
Typical Pressure
B (1 8 )
Analysis Time*
(mm) N(5 µm) N(3.5 µm) N(1.8 µm) Bar (1.8 µm)
150 12,500 21,000 32,500 >400
100 8 500 14 000 24 000 >400AnalysisTi 33%100 8,500 14,000 24,000 >400
75 6000 10,500 17,000 320
50 4 200 7 000 12 000 210
Time
PeakVolume
-33%
-50%
67%50 4,200 7,000 12,000 210
30 N.A. 4,200 6,500 126
15 N A 2 100 2 500 55
Volume -67%
-80%
-90%SolventUsage15 N.A. 2,100 2,500 55 -90%
* Reduction in analysis time compared to 150 mm column
Page 8
Note: pressure determined with 60:40 MeOH/water, 1ml/min, 4.6mm ID
Goal: Run Current Method 2X FasterTrick: ½ Column Length and Smaller Particle = ½ TimeTrick: ½ Column Length and Smaller Particle ½ Time
Rapid ResolutionStableBond SB-C18
StableBond SB-C184.6 x 150 mm, 5 μm1
1Mobile Phase: 45% 25 mM NaH2PO4, pH 3.055% MeOH
Fl R t 2 0 L/ i
StableBond SB C184.6 x 75 mm, 3.5 μm
4.6 x 150 mm, 5 μm
Flow Rate: 2.0 mL/min.Temperature: 35°CDetection: UV 254 nmSample: Cardiac Drugs
1. Diltiazem Analysis Time:5 2 i
532
53
2
2. Dipyridamole3. Nifedipine4. Lidoflazine5. Flunarizine
5.2 minAnalysis Time:11.7 min
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14Time (min)
4
0 1 2 3 4 5 6Time (min)
4
Page 9
Goal: Run Current Method 3X Faster Trick: 1/3 Column Length and Smaller Particle = 1/3 Time
Original methodZORBAX LC column Extend C18
mAU
Extend-C184.6 x 150 mm, 5 μm3 μL inj.
100
150
200
250
12 5
4
36
min0 2 4 6 8 10
0
50
2 5
mAU
80
100
120 ZORBAX RRHT column Extend C184.6 x 50 mm, 1.8 μm1 μL inj2 3
4
51 6
3x faster
-20
0
20
40
60
Mobile phase: (70:30) MeOH: 50 mM pyrrolidine buffer Flow = 1.0 mL/min, Temp. : ambient
1 μL inj.2 3 5 6
Page 10
min0 2 4 6 8 1020
How Long Can the Column Be with MeOH?System Pressure With MeOH/Water
P t f P (b )
4.6 x 50mm Column with 1.8um particles at 1 ml/min,30oC
Percentage of MeOH
Pressure (bar)
0 14210 169
Pressure curve
200
250
e 10 16920 20330 22940 2490
50
100
150
pressur
40 24950 25260 24070 216
0 20 40 60 80 100
percentage of MeOH
70 21680 18490 145100 98
•Use P of 4.6 x 50mm as baseline
•Multiply P by Lcol2 / Lcol1
•If P is lower than max P of Instrument
Page 11
100 98you can run that length
How Long Can the Column Be with ACN?System Pressure with ACN/Water
4.6 x 50mm Column with 1.8um particles at 1 ml/min,30oC
Percentage of ACN (%)
Pressure (bar)
0 14210 154
System pressure with the change ofpercentage of ACN
150
re
10 15420 15930 15640 150
0
50
100
0 50 100
Pressur
40 15050 13560 12070 104
Percentage of ACN
•Use P of 4.6 x 50mm as baseline70 10480 8890 70
•Multiply P by Lcol2 / Lcol1
•If P is lower than max P of instrument you can run that length
Page 12
100 59instrument you can run that length
More Resolution in Original Method than NeededTip: 1.8um, Very Short Column, Faster Flow 5-10X Faster p yIsocratic on instrument with 400bar pressure limit
4.6 x 250 mm, 5 μm29.65
12
3
4
Rs(1,2) = 4.8N 21848
min0 5 10 15 20 25 30
4.6 x 100 mm, 3.5 μm12 71
12
3
4
Rs(1,2) = 3.5
N=22680N=21848
.
4.6 x 30 mm, 1.8 μm min0 5 10 15 20 25 30
12.71
12
3
4
R (1 2) 3 3
N=11691
N=6568 6 30 , 8 μ1 mL/min
min0 5 10 15 20 25 30
4.15
1 2 3
4
Rs(1,2) = 3.3
N=6104
N=6568
4.6 x 30 mm, 1.8 μm2 mL/min
min0 5 10 15 20 25 30
0.5 1 1.5 2 2.52.09
2 3
4Rs(1,2) = 3.1
C l ZORBAX SB C18 M bil Ph 50% 20 M N H PO H 2 8 50% ACN Fl R t 1 L/ i T t RT
N=6460
N=6463
Page 13
Columns: ZORBAX SB-C18 Mobile Phase: 50% 20 mM NaH2PO4, pH 2.8: 50% ACN Flow Rate: 1 mL/min Temperature: RTDetection: UV 230 nm Sample: 1. Estradiol 2. Ethynylestradiol 3. Dienestrol 4. Norethindrone
Tip: Constant Linear Velocity Maintains EfficiencyTrick: Reduce Flow Rate When Reducing Column Diameter
1mAU
175
Mobile Phase: 25% methanol in 0.4% Formic Acid
75
100
125
150
ZORBAX SB-C18, 4.6 x 250 mm, 5 μm, 1 mL/min
Solvent Used: 34 mL2
3
4
min0 5 10 15 20 25 30 35
0
25
50
mAU
2 45 6
min5 10 15 20 25 30 35U
100
125
150
175
ZORBAX SB-C18, 3.0 x 100 mm, 3.5 μm, 0.425 mL/minSolvent Used: 5.7 mL, decrease of 83% (decrease in analysis time of 57%)
0
25
50
75
Page 14
min0 5 10 15 20 25 30 35
Reference for Common Column DiametersMaintain Equivalent Linear Velocity for Different Column IDsMaintain Equivalent Linear Velocity for Different Column IDs
Column Type Column ID Flow RateypAnalytical 4.6 mm 1.0 mL/min
Solvent Saver 3.0 mm 0.42 mL/min
N B 2 1 0 21 L/ iNarrowBore 2.1 mm 0.21 mL/min
MicroBore 1.0 mm 47 μL/min
Capillary 0.5 mm 12 μL/minp y μ
Capillary 0.3 mm 4.2 μL/min
Nano 0.1 mm 472 nL/min
Flow rate column 2 = (diameter column 2)2/(diameter column 1)2 x Flow rate column 1
Nano 0.075 mm 266 nL/min
Page 15
Maintain equivalent mobile phase linear velocity when changing column diameter.
Isocratic Tips and Tricks Summary• Increased isocratic speed does not necessarily require ..sub-2um particles
P ti l di t d l l th j f t i• Particle diameter and column length are major factors in ..resolving power
• Chose new shorter column to approximate original column• Chose new, shorter column to approximate original column ..efficiency; Choose length based on time savings goal
– Example 3X: 150mm, 5µm (~12,000N) ~ 50mm, 1.8µm (~12,000N)p , µ ( , ) , µ ( , )
• Usually only need to reduce column length and particle size– Not necessary to change flow, injection vol. or organic content of MP
• Altered column diameter requires change in flow and injection ..volume
Page 16
GRADIENT GRADIENT ELUTIONELUTION
Page 17
Reducing Particle SizeReduces peak width; Increases height; DOES NOT change the gradient
mAU
16001.8um vs. 5um+ 60% Resolution
1200
1400 + 160% Efficiency
800
1000 Pressure at start: 51bar, 5µmRs(4,5) = 3.14Peak Width 5σ (9) = 0.107
200
400
600
Pressure at start: 394bar, 1.8µmRs(4,5) = 5.07Peak Width 5σ (9) = 0 074
min2 4 6 80
200 Peak Width 5σ (9) 0.074
Page 18
Note: Chromatograms offset for better view
Resolution Equation for Gradient Elution Relationship of k* to p
Gradient Time, Flow and Column Dimensions
Zorbax V = π x (Col internal radius)2 x Length x 0 6
VR ≈ N k*α
Zorbax Vm = π x (Col internal radius)2 x Length x 0.6
R ≈4
kα
Δ%B = difference between initial and final % B
tg Fk* ∝
Δ%B = difference between initial and final % B ...... S = constant (≈ 4 for 100 - 500Da)
F = flow rate (mL/min)tg = gradient time (min)
S (Δ%B) Vmg g ( )
Vm = column void volume (mL)
Page 19
Expected: Use of a Longer Gradient Time Increases Gradient RetentionIncreases Gradient Retention
4
2
1,21
3
5C l ZORBAX SB C8
Gradient Time
10 min.
Gradient Time60 min.
34 5 7
Column: ZORBAX SB-C84.6 x 150 mm, 5 µm
Gradient: 20 – 60% BMobile Phase: A:H2O with 0.1%TFA, pH 2
B: AcetonitrileFlow Rate: 1 0 mL/min
8
6,7
8
6
Flow Rate: 1.0 mL/minTemperature:35°CSample: Herbicides
0 25 500 5 10 15Time (min)Time (min)
8
Page 20
Increased gradient retention improves resolution of several peak pairs – 1,2 and 4,5
Tip: A Shorter Column (smaller Vm) Also Increases Gradient Retention and RsGradient Retention and Rs
1,2
4 6 150 5
4
5
Column: ZORBAX SB-C8
4.6 x 150 mm, 5 μm 4.6 x 75 mm, 3.5 μm
2
34
5
7
8Gradient: 20 – 60% BMobile Phase: A:H2O with 0.1%TFA, pH 2
B: AcetonitrileFlow Rate: 1.0 mL/minTemperature:35°CS l H bi id
6
8
1
3
67
Sample: Herbicides
0 5 10 15Time (min)
0 5 10Time (min)
I d di t t ti I ll l ti
Page 21
Increased gradient retention; Increases overall resolution
Tip: Improve Resolution By Using Short Column Length (Vm ) for Biomolecules
300SB C8 4 6 150 5 300SB C8 4 6 50 3 5
Length (Vm ) for Biomolecules
300SB-C8, 4.6 x 150 mm, 5µm 300SB-C8, 4.6 x 50 mm, 3.5µm
24 5
6 45
6
1
2
3
5
7 8
9
101
23
5
7 8
9
10
M bil Ph A 95% W t 5 % ACN 0 1% TFA S l 1 Gl T 6 L E k
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
Mobile Phase: A: 95% Water : 5 % ACN, 0.1% TFAB: 5% Water : 95% ACN, 0.085% TFAGradient: 10-60% B in 30 min.
Flow Rate: 1.0 mL / min.Temperature: Ambient
Sample: 1. Gly-Tyr 6. Leu-Enk2. Val-Tyr-Val 7. Angiotensin II3. [Gln11] Amyloid-β- 8. Kinetensin
Protein Fragm 1-16 9. RNase4. (TYR8) Bradykinin 10. Insulin (Eq.)5 M t E k
Page 22
5. Met-Enk
Tip: Maintain k* To Keep Relative Peak Position in a Chromatogram Unchanged While Reducing Timea Chromatogram Unchanged While Reducing Time
V - Column length Decrease in tG or F
Any Decrease in Can be Offset by a Proportional
Vm - Column length
V - Column (id)
G
Decrease in tG or FVm Column (id) Decrease in tG or F
k* ∝tG • F
S • Δ%B • Vm
Page 23
Trick: Change Vm and tg by Same ProportionTwo Chromatograms Both Having the Same Gradient Steepness
Column: Rapid Resolution
Sample: 1. Tebuthiuron 2. Prometon 3. Prometryne 4. Atrazine 5. Bentazon 6. Propazine 7. Propanil 8. Metolachlor
o C o atog a s ot a g t e Sa e G ad e t Steep ess
4
5
Column: StableBond SB-C84.6 x 150 mm, 5 μm
Gradient Time: 30 min.
Flow Rate: 1.0 mL/min4
5
Column: Rapid Resolution StableBond SB-C84.6 x 75 mm, 3.5 μm
Gradient Time: 15 min.
Flow Rate: 1 0 mL/min
8
8
Flow Rate: 1.0 mL/min
1
2
3AnalysisTime: 24 min 6
7
1
2
3
6
AnalysisTime: 12 min
0 5 10 15 20 25
77
Page 24
Time (min)
0 5 10 15 20 25
Time (min)
0 5 10 15
001784S1.PPT
Very Fast LC on Conventional 1100 HPLC Th I t tG1379 DegasserG1311 Quaternary pump
The Instrument
G1313A ALS autosamplerG1316A column compartmentG1314A VWD (standard cell G1314-60086, 10mm, 14uL)
AcetophenoneThe Sample
pDiethyl phthalateBenzophenoneButyrophenoneValerophenoneHexanophenoneHeptanophenoneOctanophenone
Page 25
Octanophenone
Page 26
Tip: Shorten Column & Gradient Time by Same FactorFactor1/3 Column Length- 1/3 Gradient TimeRRHT Column – 4.6 x 50mm, 1.8µm, SB-C18
VWD1A Wavelength=246nm(D:\SAMPLETEST\RRHT1100\HDS20070809172525\070809SBC180009D)Flow Rate 1.0 ml/min
mAU
120
140
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS 2007-08-09 17-25-25\070809SBC180009.D)
1.1
76
2.2
5 4
3.3
57 Injection Volume 5uLTemperature 30°CWavelength 246nmSample rate 13 74 Hz
60
80
100
2.00
4
2.4
64
2.8
11
3.8
71
4.3
43
Sample rate 13.74 Hz
Time (min) % Acetonitrile
0
20
40
0 503.33 904.5 904 53 50
min0 1 2 3 4 5 6
0 4.53 505 50
Initial Pressure: 132 barFinal Pressure: 74 bar
Page 27
Final Pressure: 74 bar
The Comparison of Chromatogram with the Same RRHT column in 1100 and 1200 SLwith the Same RRHT column in 1100 and 1200 SL
VWD1 A Wavelength=246 nm (D:\SAMPLE TEST\RRHT 1100\HDS 2007 08 09 17 25 25\070809SBC180008 D)
Overlay the two Chromatograms
Norm.
120
140
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS 2007-08-09 17-25-25\070809SBC180008.D) 1
.179
2.2
57
3.3
61
*DAD1 A, Sig=246,4 Ref=360,100 (WORKSHOP-TAIWAN\070815SBC180003.D)
80
100
2.4
68
6
40
60
2.0
07
2.8
1
3.8
74
4.3
43
min1 1.5 2 2.5 3 3.5 4 4.5 5
0
20
Page 28
The red one is 1200, the blue one is 1100.
Page 29
Gradient Tips and Tricks Summaryt Ftg F
S (Δ%B) Vm
k* ∝
Gradient Retention Relationship Formula Is the Key to:
( ) m
• Improving Resolution– Longer gradient time (tg)– Smaller column volume (Vm), length or ID– Keeping all other parameters constant
•Increasing Speed Without Losing Resolution•Increasing Speed Without Losing Resolution– Shorter column with proportionally shorter gradient time– Shorter gradient time with proportionally faster flow rate
Page 30
Shorter gradient time with proportionally faster flow rate
Tip: Method Translator Makes Changes EasyTrick: Let Agilent Method Translator Do the Math
Injection Volume Conversion
Basic Mode for Easy Transfer of Conventional Method to RRLC
Detector Settings recommendation
Gradient and Isocratic Method Conversion (auto-detected))
Page 31
Detailed OutputDetailed Input
Agilent Method Translator – Advanced ModeMore Detaled Information, But Still Easy to Use
pp
O i i l M th d N M th d
Page 32
Original Method New Method
Analysis of impurities of an active pharmaceutical ingredient by Does it work? - Example
conventional HPLC (4.6mm ID x 250 mm, 5.0 µm):mAU
OH
HN
CH3CH3
30
40 OH
OCH3
Main Compound
CH3CH3
20OH
HN
3C3
OCH3Impurity A
NCH3CH3CHCH
N33
H
NCH3CH3
H
10
Impurity A
O CH3
Br
Bromanisole
OCH3
Impurity B
OCH3
Impurity C
OH
OH
Impurity D
i0 2 5 5 7 5 10 12 5 15 17 5 20
0
Page 33
min0 2.5 5 7.5 10 12.5 15 17.5 20
Converting to a 4.6 mm ID x 100 mm, 1.8µm column:
Page 34
Does it work? Yes!mAU
30
40 Conventional HPLC
0
10
20
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU14
Resolution optimized 1.8µm
6
8
10
12
-2
0
2
4
Added Benefit of Increased Sensitivity!
Page 35
min0 1 2 3 4 5 6 7 82
mAU Conventional HPLC mAU35
Does it work?
30
40
25
30
mAU
20
2015
20
10
15
0
10
0
5
10
0
5
4.6 mmID x 250 mm, 5.0µm Zorbax SB C18
min0 2.5 5 7.5 10 12.5 15 17.5 20
0
4.6 mmID x 100 mm, 1.8µm Zorbax SB C18
min0 1 2 3 4 5 6 7 8
0
min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
0
4.6 mmID x 100 mm, 1.8µm Zorbax SB C18 Speed Optimized0.00 min 5% B
20.00 min 90% B23.00 min 90% B23.01 min 5% B30 00 i 5% B
0.00 min 5% B8.00 min 90% B9.20 min 90% B9.21 min 5% B
12 00 i 5% B
Simple Conversion0.00 min 5% B4.33 min 90% B4.98 min 90% B4.99 min 5% B6 5 i 5% B
Speed Optimized
Page 36
30.00 min 5% B12.00 min 5% B6.5 min 5% B
ZORBAX Selectivity Choices Options for Improved Speed and ResolutionOptions for Improved Speed and Resolution
Reversed-Phase Chromatography Specialty d t
Extend-C18Bidentate-C18
StableBondDiisopropyl
Diisobutyl bonding
Eclipse XDBDimethyl bonding
Eclipse PlusDimethyl bonding
products
Double endcapping
SB-C18Diisobutyl C18
Diisobutyl bonding
Rx-C18
D gy g
Double endcapping
Eclipse XDB-C18SB-CNDiisopropyl-CN
D gy g
Double endcapping
Eclipse Plus C18Diisobutyl-C18
Rx/SB-C8Diisopropyl-C8
SB-C3Triisopropyl
DimethyloctadecylsilaneEclipse XDB-C8
Bonus-RPE b dd d id
Diisopropyl CN
Eclipse Plus C8
E li Pl
Eclipse PAH
SB-PhenylDiisopropyl-
Phenyl
SB-AQDiisopropyl
Eclipse XDB-Phenyl
E li XDB CN
Embedded amideDiisopropyl-C14
Triple endcappedEclipse Plus-Phenyl-Hexyl
Page 37
Eclipse PAHEclipse XDB-CN
Tip: Bonded Phase Can Change SelectivityImprove Peak Shape and Shorten HPLC Assay TimeImprove Peak Shape and Shorten HPLC Assay Time
• Each ZORBAX phase available in matching 1.8µm,3 5µm and 5µm choices most in 7µm!3.5µm and 5µm choices, most in 7µm!
• Over 140 RRHT,1.8um 600 bar column choices available
• 14 Different Column Chemistries – 13 bonded phases pand silica for HILIC use
• 7 column lengths (250* 150 100 75 50 30 and 20• 7 column lengths (250 , 150, 100, 75, 50, 30 and 20 mm long) * 1.8µm as custom column
3 i l di (4 6 3 0 2 1 d P ID)
Page 38
• 3 internal diameters (4.6, 3.0, 2.1 mm and Prep ID)
Tip: C8 Often Yields Same Peak Order in < Time Than C18 Trick: Evaluate Different Phases with Same MP
1. Oxybenzone
RRHT Eclipse Plus C18, 4.6 x 50mm, 1.8 umy
2. Internal Std.3. Octylmethoxycinnamate
N: 8800Pw: 0 014 min
N: 11600Pw: 0 047 min
N: 11800Pw: 0 093 min
247 bar
Rapid Resolution Eclipse Plus C8, 4.6 x 50mm, 3.5 um
Pw: 0.014 min.TF: 1.20
Pw: 0.047 min.TF: 1.06
Pw: 0.093 min.TF: 1.02
88 barmin0 1 2 3 4)
Conditions:Mobile Phase.: Water: Acetonitrile (30:70)Flow Rate: 2.0 mL/min. Detection: UV 230nm Temperature: 30 °C
2.2 min.N: 6600Pw: 0.037 min.TF: 0.99
N: 6300Pw: 0.061 min.
N: 5000Pw: 0.019 min.TF: 1.09
Temperature: 30 CSample: Lip balm extract in ACN(melted at 100°C ACN , cooled and 0.45 um filtered)
TF: 0.98
min0 1 2 3 4Less retention can save significant time – the C8 is a good choice here.The RRHT column is delivering the efficiency and resolution expected but the C8 bonded phase may be
Page 39
Group/Presentation TitleAgilent Restricted
Month ##, 200X
The RRHT column is delivering the efficiency and resolution expected, but the C8 bonded phase may be the best choice.
Tip: Polar Phases Can Save Analysis TimeTrick: Evaluate Non-Polar and Polar Phases with Same MP
Eclipse Plus Phenyl Hexyl
min0 2 4 6 8 10
Elution order reversal between Phenyl-Hexyl and Alkyl chains
Eclipse Plus C8
min0 2 4 6 8 10
y y y
Eclipse Plus C18
Mobile Phase 40 % ACN 60 % 25 mM Sodium Phosphate Buffer pH 2.4 Flow Rate: 1.5 ml/min 4.6 x 50mm UV 210 nm 2µl Elution order for Eclipse Plus Phenyl Hexyl: (1) Piroxicam, (2) Sulindac,(3) Tolmetin, (4) Naproxen, (5) Ibuprofen, (6)
min0 2 4 6 8 10
Page 40
Group/Presentation TitleAgilent Restricted
Month ##, 200X
µ p y y ( ) , ( ) ,( ) , ( ) p , ( ) p , ( )Diclofenac, (7) Celebrex (equal portions of approximately 1 mg/ml solutions
Tip: Higher Temperature Can Improve Speed and ResolutionSpeed and Resolution
Higher Temperature should always be considered
as a parameter during speed optimizationas a parameter during speed optimization
• Provides more rapid mass transfer:Improves Efficiency enhances resolution– Improves Efficiency – enhances resolution
– Decreases analysis time – faster separations with no loss in resolution
• Decreases mobile phase viscosityDecreases mobile phase viscosity– Lowers backpressure – allows for higher flow rates, faster separations,
greater efficiency
• Can change selectivity – optimize resolution
• Not necessary to go to the limit; Midrange offers benefits
Page 41
Tip: Temperature Changes Can Alter SelectivityTrick: Optimize Col Temp For Best Speed/Rs
20°CSalicylic acid
min0 1 2 3 4
30°C
0 1 2
0 1 2 345
3 4
Salicylic acidCoelution
40°C
0 1 2
0 1 2 345
Salicylic acid
60°C
0 1 2 345
0 1 2 345
Salicylic acid Column: RRHT SB-C184.6 x 50mm, 1.8um
90°C
0 1 2 345
345
Salicylic acid Increased T Decreases Run Time, Changes Selectivity and Sharpens Peaks
Improving Resolution
Page 42
0 1 2 345
Tips on When You Should Move to the Higher Pressure and Performance 1200 SL (RRLC) SystemPressure and Performance 1200 SL (RRLC) System • Very fast methods with high velocity mobile phase that increase backpressure above 400barp
• Resolution of difficult samples require long columns with 1.8µm particles (>400bar backpressure)
• Labs required to run both traditional and new, higher performance LC methods
• Need faster Data Acquisition for narrow, high efficiency peaks generated in higher speed methods
Page 43
Tip: 1.8µm Particle Increases Resolution; How?Trick: Substitute 1.8µm Particles in Same Length Column
Conditions for bothmAU 5Agilent 1100 Zorbax 2.1x150mm Conditions for both experiments
Pumps• Solvent A: H2O + 0.1% TFA
Solvent B: ACN + 0 1% TFA
mAU
250
300
350
17.1
36
15.9
67
59
15.5
55
15.0
65Agilent 1100 Zorbax 2.1x150mm SB C-18, 5µm
Solvent B: ACN + 0.1% TFA• Gradient: 10% to 95% ACN
in 40min, hold for 1min• Flow Rate: 0.4ml/minAutosamplers
100
150
200
15.4
1115.1
5Autosamplers• Injection volume: 3µlThermostatted Column
Comp.T t 50°C
Agilent 1200 RRLC250 2
mAU
079 Zorbax 2.1x150mm
SB C 18 1 8
min13 14 15 16 17 18
50
• Temperature: 50°CDetectors• DAD 2µl cell and 20Hz,
220nm, 150
200
250
2415.1
64 16.7
4615.4
7 215
.79015
. 0 SB C-18, 1.8µm
Ref: Appl. Note 5989-4506 by Edgar Naegele
50
100 15.3
2114
.937
14.7
87
Page 44
min13 14 15 16 17 18
Tip: Small Particles Improve Detection of Low Level Impurities
mAU
2
2.5
DAD1 A, Sig=254,4 Ref =of f (051119A\SIG10003.D) Column:150x4.6 mm
5µm
Pressure: 93 bar
N: 8213
4.6 x 150, 5um93 barN = 7259R S 1 15 20000
25000
30000
pdN 1
∝
p
0 5
0
0.5
1
1.5 Height: 1.25 mAU
S/N: 42.3
Rs = 1.15
tr = 14.9 min (1st epimer)
Nptp: 2.4 10-2 mAU
R_S = 1.15S/N = 42 Height = 1.25Noise = 24uAU
N
5000
10000
15000
20000 pd
min0 5 10 15 20
-1
-0.5
Norm.
DAD1 A, Sig=254,4 Ref=off (051007D\LC_X000001.D)
Norm.
DAD1 A, Sig=254,4 Ref=off (051007D\LC_X000001.D)Norm.
DAD1 A, Sig=254,4 Ref=off (051004D\LC_J0002.D)
Norm.
DAD1 A, Sig=254,4 Ref=off (051004D\LC_J0002.D)
Norm.
DAD1 A, Sig=254,4 Ref=off (051007D\LC_X000001.D)
Norm.
DAD1 A, Sig=254,4 Ref=off (051007D\LC_X000001.D)Norm.
DAD1 A, Sig=254,4 Ref=off (051004D\LC_J0002.D)
Norm.
DAD1 A, Sig=254,4 Ref=off (051004D\LC_J0002.D)1/dp
0.2 0.3 0.4 0.5 0.65000
Column:150 x 4.6 mm 1.8µm
Pressure: 490 bar
N: 28669
Height:1.78
S/N: 43.61.5
2
2.5
3Column:150 x 4.6 mm 1.8µm
Pressure: 490 bar
N: 28669
Height:1.78
S/N: 43.61.5
2
2.5
3Column:150x4.6 mm
3.5µm
Pressure: 165 bar
N: 14862
Height:1 34 mAU1
1.5
2
2.5 Column:150x4.6 mm
3.5µm
Pressure: 165 bar
N: 14862
Height:1 34 mAU1
1.5
2
2.5 Column:150 x 4.6 mm 1.8µm
Pressure: 490 bar
N: 28669
Height:1.78
S/N: 43.61.5
2
2.5
3Column:150 x 4.6 mm 1.8µm
Pressure: 490 bar
N: 28669
Height:1.78
S/N: 43.61.5
2
2.5
3Column:150x4.6 mm
3.5µm
Pressure: 165 bar
N: 14862
Height:1 34 mAU1
1.5
2
2.5 Column:150x4.6 mm
3.5µm
Pressure: 165 bar
N: 14862
Height:1 34 mAU1
1.5
2
2.5 4.6 x 150, 3.5um165 barN = 14862R_S = 1.37
4.6 x 150, 1.8um490 barN = 28669 R_S = 1.80 (+57%)S/N: 43.6
Rs = 1.80
tr = 17.2 (1st epimer)
Nptp: 3 10-2
-1
-0.5
0
0.5
1S/N: 43.6
Rs = 1.80
tr = 17.2 (1st epimer)
Nptp: 3 10-2
-1
-0.5
0
0.5
1Height:1,34 mAU
S/N: 50.7
Rs = 1.37
tr = 15.3 min (1st epimer)
Nptp: 2 10-2 mAU
-1
-0.5
0
0.5
Height:1,34 mAU
S/N: 50.7
Rs = 1.37
tr = 15.3 min (1st epimer)
Nptp: 2 10-2 mAU
-1
-0.5
0
0.5
S/N: 43.6
Rs = 1.80
tr = 17.2 (1st epimer)
Nptp: 3 10-2
-1
-0.5
0
0.5
1S/N: 43.6
Rs = 1.80
tr = 17.2 (1st epimer)
Nptp: 3 10-2
-1
-0.5
0
0.5
1Height:1,34 mAU
S/N: 50.7
Rs = 1.37
tr = 15.3 min (1st epimer)
Nptp: 2 10-2 mAU
-1
-0.5
0
0.5
Height:1,34 mAU
S/N: 50.7
Rs = 1.37
tr = 15.3 min (1st epimer)
Nptp: 2 10-2 mAU
-1
-0.5
0
0.5
_S/N = 50 Height = 1.34Noise = 20uAU
_ ( )S/N = 44 Height = 1.80Noise = 30uAU
Page 45
min101
min101
min0 5 10 15 201
min0 5 10 15 201
min101
min101
min0 5 10 15 201
min0 5 10 15 201
Particles Reveal More Information and Improve Detection and IntegrationDetection and Integration
7 Impurities7 Impurities4 Impurities
Customer Sample, Translation of Isocratic Impurity Methods, Zoom Critical Time Range (t = 7min)
7 Impurities
All 7 Baseline Separated!
7 Impurities
6 Not Baseline Separated!
4 Impurities
2 Not Baseline Separated!
4.6 x 150, 1.8μm490 barN = 28669
4.6 x 150, 3.5μm165 barN = 14862
4.6 x 150, 5μm93 barN = 7259
R_S = 1.80 (+57%)S/N = 44
R_S = 1.37S/N = 50
R_S = 1.15S/N = 42
Up to 60% higher resolutionwithout loss in sensitivity
Page 46
without loss in sensitivity
Example of What Is Possible!Shorter Column, Smaller Particle, > Flow Rate, High Temp
F= 1.20ml/minT = 40°CAnalysis Time = 11min
High Resolution:4.6mm x 150mm 5.0µm
, , , g pEasy!
Solvent Cons. = 13.2ml
min0 2 4 6 8 10 12
F = 4.80ml/minT = 40°CAnalysis Time = 1 05min
High Speed:Easy!
Analysis Time = 1.05minSolvent Cons. = 5.1ml
4.6mm x 50mm 5.0µm
min0 0.2 0.4 0.6 0.8 1
F= 1.00ml/minEasy!
T = 40°CAnalysis Time = 1.1minSolvent Cons. = 1.1ml
High Speed & Resolution:2.1mm 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°CAnalysis Time: 0.4minSolvent Cons. = 1.0ml
PWHH = 197msec
> 20x faster !
Easy, But You Will Need > 400bar!
Page 47
min0.2 0.4 0.6 0.8 10
Tip: Higher Data Improves Response and Rs Trick: Optimize Data Rate for Accurate Profile
PW=0 30se 80Hz versus 10Hz Data Rate
Trick: Optimize Data Rate for Accurate Profile
80Hz
PW=0.30sec
80Hz versus 10Hz Data Rate• Peak Width: – 55%• Resolution: + 90%• Peak Capacity: + 120%• App Column Eff : + 260%
Data Rate
Peak Width
Resolution Peak Capacity
80 Hz 0.300 2.25 6040Hz
PW=0.33sec• App. Column Eff.: + 260%
40 Hz 0.329 2.05 55
20 Hz 0.416 1.71 45
10 Hz 0.666 1.17 29
5 Hz 1 236 0 67 16
20Hz
PW=0.42sec
PW=0 67sec 5 Hz 1.236 0.67 16
min0 1 0 2 0 3 0 4 0 50
10HzPW 0.67sec
5HzPW=1.24sec
Sample: Phenones Test MixColumn: Zorbax SB-C18, 4.6x30, 1.8umGradient:: 50-100%ACN in 0.3minFlow Rate: 5ml/min
Page 48
min0.1 0.2 0.3 0.4 0.50 Flow Rate: 5ml/min
Moving From Conventional LC to RRLC:“Doesn’t this
Moving to RRLC is simple and easy !Moving to RRLC is simple and easy !
• Same software (e g ChemStation EZChrom)
Doesn t this cost a fortune ?”
• Same software (e.g. ChemStation, EZChrom)
No additional training effort
• Limit capital investment by modular step-wise upgradep y p pg
Leverage existing 1100 modules, existing method
and stationary phase
• Configurable delay volume in 1200 SL pump
Easy to run legacy LC methods too
• Simple LC to RRLC method translator
No tedious method development“No - not at all !”
Page 49
Stepwise Scale-up to Rapid Resolution LCFrom 1100 to 1200 RRLC in two steps -Add Wh t Y N d Wh Y N d It!Add What You Need When You Need It!
Actual: 1100
Quat System
Step 1: 1100/1200
„Bin SL“ System
µ-Degasser
Step 2: 1200
Rapid Resolution System
Bin Pump SL
u-Degasser
Quat System
ALS
Quat Pump
Degasser
h-ALS SL
TCC
Bin Pump SL h-ALS SL
TCC SL
DAD SL
TCC
VWD/MWD/DAD
VWD/MWD/DAD+ Speed
+ Resolution+ Sensitivity
+ MS-Robustness+ Data Security & Traceability
+ Resolution+ Speed
+ MS-Compatibility
> 5 min> 6 min
> 1.5min> 2min
Analysis TimeCycle times
> 0.2min> 0.4min
y y+ Qualification (Degasser, ACE)+ Compatible with conv. HPLC
+ MS Compatibility+ Solvent Saving
+ Compatible with conv. HPLC
> 3 sec5 - 12,000
4.6 mm50 mm
0.2 - 10ml/min80 C
> 1.5 sec5 - 30,000
2.1 - 4.6 mm50 - 150 mm
0.05 - 5 ml/min80 C
Peak WidthN
Column IDColumn Length
Flow ratesT t
> 0.2 sec5 - 60,000
2.1 - 4.6 mm20 - 150mm
0.05 – 5 ml/min
Page 50
80 C400bar
80 C600 bar
TemperaturePressure
100 C600bar
SUMMARY
• Increasing speed of HPLC separations need not be difficult or costlycostly• Changes in columns and equipment depend on your goals• Majority of time saved will be based on column and particleMajority of time saved will be based on column and particle size• Isocratic and gradient 2X , 3X and 5X times savings are easy • Greater time savings may require instrument modifications• 10-20X increase in speed may require instrument upgrade• Long columns with 1.8µm particles may require >400 bar pump and autosampler capability
Page 51