The One Minute Chromatographer - Agilent · Agilent ZORBAX RRHD SB-C18, 2.1 x 100mm, 1.8 µm Sub 1 Minute Separations with RRHD Columns . Page 3 Longitudinal ... ±25% (FDA ORA-LAB
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Page 1
The One Minute
Chromatographer
Agilent Technologies, Inc.
2013
H2O (0.05% Formic acid) / 2-98 % Acetonitrile
T = 80°C
V_i = 0.5 µl
WL = 210 nm
DR = 80 Hz
F = 2 ml/min
P = 1085 bar
Agilent ZORBAX RRHD SB-C18, 2.1 x 100mm, 1.8 µm
Sub 1 Minute Separations with RRHD Columns
Page 3
Longitudinal
diffusion
Smaller Particles Reduce In–Column Dispersion
Increased Efficiency P
late
Heig
ht
H
Linear Velocity u
Eddy Diffusion
Sum Curve: van-Deemter
Resistance to Mass Transfer
H = A + B/u + C u
Large
Particle
Small
Particle
u opt
H min
Sub 2um Particle
Superficially Porous Particle
Two Advances For Better Mass Transfer
SOLID
CORE
Poroshell 120 Columns for HPLC and UHPLC:
• 80-90% efficiency of sub 2um
• At ~40-50% lower pressure
• 2X efficiency of 3.5um (totally porous)
• A 2.7um particle size
• A 2um frit to reduce clogging
• A 600 bar pressure limit
• The particle has a solid core (1.7um) and
porous outer layer with a 0.5um diffusion
path
July 17, 2013
Confidentiality Label
5
Poroshell 120 columns have:
1.7um
0.5um
0.5um
Page 6
Poroshell 120 – Several Different Phases
EC-C18
EC-C8
EC-CN
Stablebond C18
Stablebond C 8
Stablebond Aq
Bonus RP
HILIC
Phenyl Hexyl
Van Deemter Redux
7
van Deemter
0
0.0005
0.001
0.0015
0.002
0.0025
0.003
0 0.5 1 1.5 2 2.5 3 3.5
Flow rate (mL/min)
HE
TP
(cm
)
Eclipse Plus C18, 3.5 µm
Eclipse Plus C18, 1.8 µm
Poroshell 120 C18, 2.6um
Poroshell 120 C18, 2.6 µm
Poroshell 120 C18, 2.7 µm
High Peak Capacity Maximizes Resolution
with Complex Samples – 1.8u Particles
High Peak Capacity Maximizes Resolution with Complex Samples – Poroshell 120
Page 10
Smaller Particles Improve
Detection and Integration
4.6 x 150, 1.8μm
490 bar
7 Impurities
All 7 Baseline
Separated!
4.6 x 150, 3.5μm
165 bar
7 Impurities
6 Not Baseline
Separated!
4.6 x 150, 5μm
93 bar
4 Impurities
2 Not Baseline
Separated!
Customer Sample, Impurity Method
What is Sensitivity?
Limit of detection (LOD):
“The lowest concentration, or smallest mass flow, which can be distinguished
from the noise by a certain predefined probability (Signal/Noise).”
Noise
Signal
hSignal = 3(2) x hNoise
Limit of detection (LOD):
Page 11
Analysis of 15 Analgesic Compounds Same Method for all Three Columns
ZORBAX Eclipse Plus C18, 3 x 100 mm, 3.5 µm
ZORBAX RRHD Eclipse Plus C18, 3 x 100 mm, 1.8 µm
Poroshell 120 EC-C18, 3 x 100 mm, 2.7 µm
Ibuprofen:
PW1/2=0.014
S/N=182
nc=43
Ibuprofen:
PW1/2=0.012
S/N=353
nc=54
Ibuprofen:
PW1/2=0.012
S/N=256
nc=56
2 min
June, 2011
Comparison of 4.6 x 250 mm 5 um to
Poroshell 120 EC-C18 4.6 x 100 mm, 2.7um
min 5 10 15 20 25 30
mAU
0
20
40
60
80
100
9.7
12
11.1
16
11.5
96
12.6
74
15.2
48
16.1
51
16.4
35
20.6
87
23.0
76
29.2
90
min 5 10 15 20 25 30
mAU
0
50
100
150
200
250
1.7
19
2.1
89
2.3
11
2.6
06
3.8
67
4.4
37 4.5
58
5.4
50
5.9
20
7.0
37
325 bar
110 bar
Sulfadiazine,
Sulfathiazole
Sulfapyridine
Sulfamerazine,
Sulfamethazine,
Sulfamethazole,
Sulfamethoxypyridazine,
Sulfachloropyridazine
Sulfamethoxazole,
Sulfadimethoxine
Time %B
0 8
33 33
34 33
Column: Eclipse Plus C18
4.6 x 250mm, 5um
Flow Rate: 1 mL/min
Mobile Phase:
A: 0.1% formic acid in Water
B: 0.1% formic acid in ACN
Time %B
0 8
12 33
13.2 33
Column: 4.6 x 100mm
Poroshell 120 EC-C18,
2.7um
Flow Rate: 1 mL/min
Expand High Speed Chromatograms for True
Comparison to Slower Separation
min 5 10 15 20 25 30
mAU
0
20
40
60
80
100
9.7
12
11.1
16
11.5
96
12.6
74
15.2
48
16.1
51
16.4
35
20.6
87
23.0
76
29.2
90
110 bar
5
mAU
0
50
100
150
200
250
1.7
19
2.1
89
2.3
11
2.6
06
3.8
67
4.4
37 4.5
58
5.4
50
5.9
20
7.0
37
325 bar
min
See conditions previous slide
Page 15
Rapid Resolution LC Ultra Fast, Resolution Maintained, Sensitivity Improved
Optimized Gradient
Time (min)
1
2
4
5
3
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Column: Rapid Resolution Eclipse XDB-C8,
4.6 x 50 mm, 3.5 mm
Mobile Phase: A: 55% 25 mM Na2HPO4, pH 3
B: 45% MeOH
Gradient: 42 – 90% B in 2 min
Flow Rate: 3 mL/min
Temperature: 35°C
Sample: Cardiac Drugs
1. Diltiazem
2. Dipyridamole
3. Nifedipine
4. Lidoflazine
5. Flunarizine 480 Samples/day
Run Time 1.8 min
Equilibration 1 min
Time
Total Analysis 2.8 min
Time
Page 16
US. Pharmacopeia 621
Column length: ± 70 %
Column internal diameter: ± 25 %
Column material particle size: Reduction of up to 50 %, no increase
Flow rate: ± 50 %
Injection volume: Changes are allowed as long as system suitability
testing (SST) criteria are met.
Column temperature: ± 10 %
pH of mobile phase: ± 0.2
UV wavelength: no change outside manufacturer specifications
Concentration of salts in buffer: ± 10 %
Composition of mobile phase (adjustment of the minor component is
allowed ±30 % or ± 10 % absolute whichever is smaller)
USP and FDA Method Adjustment Criteria Method Adjustment Criteria for Column Dimensions
Parameter Maximum Specifications Comments/Examples
Column Length ± 70% 250mm 75mm
150mm 50mm
Column Internal Diameter
±25% (FDA ORA-LAB 5.4.5*)
USP – ± 25% Column ID can be adjusted provided
linear velocity is constant**
4.6 mm 3.0 mm (-35%)
4.6 mm 2.1 mm (-54%)
3.0 mm 2.1 mm (-30%)
Flow Rate ±50%
Injection Volume
Reduce as much as needed –
must still meet detection limits and
precision
If you change to a smaller/ shorter
column make the appropriate
change in injection volume
Particle Size Reduce by up to 50%
(can not be increased)
You can change column length
and particle size to keep Rs same
5um 3.5um (-30%)
5um 2.7um (-46%)
*Changed on 2/24/09 - For the current and official copy, go to
http://web.ora.fda.gov/dfs/policies/manuals/default.html
** USP 30 Second Supplement Revisions, PF34(5), in process expected to be final Dec 2009
See Stimuli article in Pharmacopeial Forum 2009; 35(6)
Page 17
Page 18
USP Method-Naproxen
Page 19
Naproxen 100 mm column
Page 20
NAPROXEN 50 mm Column
Agilent 1200 Rapid Resolution System – Speed
Conventional LC → UFLC → RRLC
Page 21
F= 1.20ml/min
T = 40°C
Analysis Time = 11min
Solvent Cons. = 13.2ml
High Resolution:
4.6mm x 150mm 5.0µm
min 0 2 4 6 8 10 12
F = 4.80ml/min
T = 40°C
Analysis Time = 1.05min
Solvent Cons. = 5.1ml
High Speed:
4.6mm x 50mm 5.0µm
min 0 0.2 0.4 0.6 0.8 1
F= 1.00ml/min
T = 40°C
Analysis Time = 1.1min
Solvent Cons. = 1.1ml
High Speed & Resolution:
2.1mm x 50mm 1.8µm
min 0.2 0.4 0.6 0.8 1 0
Max Speed at T = 95oC
2.1mm x 50mm 1.8um
F= 2.40ml/min
T = 95°C
Analysis Time: 0.4min
Solvent Cons. = 1.0ml
PWHH = 197msec
min 0.2 0.4 0.6 0.8 1 0
> 20x faster !
Group/Presentation Title
Agilent Restricted
Month ##, 200X
0 10 20 30 40
Changing Gradient Time to Affect Retention (k*) and Resolution
Time (min)
100% B
100% B
100% B
100% B
tg= 40
tg= 20
tg= 10
tg= 5
000995P1.PPT
1/k* = gradient steepness = b
tg F
S D%B Vm
k* =
DF = change in volume fraction of B
solvent
S = constant
F = flow rate (mL/min.)
tg = gradient time (min.)
Vm = column void volume (mL)
0% B
0% B
0% B
0% B
• S 4–5 for small molecules
• 10 < S < 1000 for peptides and proteins
Page 23
Very Fast LC on Conventional 1100 HPLC
G1379 Degasser
G1311 Quaternary pump
G1313A ALS autosampler
G1316A column compartment
G1314A VWD (standard cell G1314-60086, 10mm, 14uL)
Acetophenone
Diethyl phthalate
Benzophenone
Butyrophenone
Valerophenone
Hexanophenone
Heptanophenone
Octanophenone
The Sample
The Instrument
Page 24
min0 2 4 6 8 10 12 14 16 18
mAU
0
50
100
150
200
250
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\070809SBC180003.D)
1.4
74
3.3
23
5.0
64
5.6
51
6.1
14
6.9
64
8.3
37
9.6
90
10.9
82
Conventional Column - 4.6 x 150mm, 5µm, SB-C18
Flow Rate 1.0 ml/min
Injection Volume 15uL
Temperature 30°C
Wavelength 246nm
Sample rate 2.5 Hz
Time (min) % Acetonitrile
0 50
10 90
13.5 90
13.6 50
15 50
Initial Pressure: 69 bar
Final Pressure: 38 bar
Page 25
Shorten Column and Gradient Time by Same Factor 1/3 Column Length- 1/3 Gradient Time
RRHT Column – 4.6 x 50mm, 1.8µm, SB-C18
min0 1 2 3 4 5 6
mAU
0
20
40
60
80
100
120
140
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS 2007-08-09 17-25-25\070809SBC180009.D)
1.1
76
2.0
04
2.2
54
2.4
64
2.8
11
3.3
57
3.8
71
4.3
43
Flow Rate 1.0 ml/min
Injection Volume 5uL
Temperature 30°C
Wavelength 246nm
Sample rate 13.74 Hz
Time (min) % Acetonitrile
0 50
3.33 90
4.5 90
4.53 50
5 50
Initial Pressure: 132 bar
Final Pressure: 74 bar
Page 26
Increase Column Flow-Reduce Gradient Time Double Flow (2mL/min) – ½ Gradient Time
RRHT 4.6 x 50mm, 1.8µm, SB-C18
min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
mAU
0
20
40
60
80
100
120
140
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS 2007-08-10 08-22-16\070810SBC180004.D) 0
.587
1.0
20
1.1
46
1.2
60
1.4
37
1.7
15
1.9
70
2.2
01
Flow Rate 2.0 ml/min
Injection Volume 5uL
Temperature 30°C
Wavelength 246nm
Sample rate 13.74 Hz
Time (min) % Acetonitrile
0 50
1.67 90
2.25 90
2.27 50
3.34 50
Initial Pressure: 266 bar
Final Pressure: 146 bar
• Flow rate vs. Gradient time vs.
Peak capacity
• For small molecules(MW < ~1000)
• Different Column Lengths
• Broken lines are isobar (800 bar)
What Length Column Yields Highest Peak Capacity?
50mm
150mm
100mm
Shorter Columns with Fast Gradients
Yield Higher Peak Capacity
50mm
150mm
100mm
Shorter Gradient (5 min)
Peak Capacity:
• 258 for 50 mm
• 240 for 100 mm
• 221 for 150 mm
Long Gradient (40 min)
Peak Capacity:
• 422 for 50 mm
• 510 for 100 mm
• 525 for 150 mm
Longer Columns with Long Gradient Times
Yield Greater Peak Capacity
50mm
150mm
100mm
Page 30
Constant Particle Size, Grad Time
More Resolution
Group/Presentation Title
Agilent Restricted
Month ##, 200X Page 30
min 0 2 4 6 8 10 12 14 16 18
mAU
0
50
100
150
200
min 0 2 4 6 8 10 12 14 16 18
mAU
0
50
100
150
200
min 0 2 4 6 8 10 12 14 16 18
mAU
0
50
100
150
200
RRHD SB-C18 2.1 x 50 mm, 1.8um
Pmax=366 bar
nc = 424
RRHD SB-C18 2.1 x 100 mm , 1.8um
Pmax=595 bar
nc = 485
RRHD SB-C18 2.1 x 150 mm, 1.8um
Pmax=768 bar
nc = 589
Rs: 2.40
Rs: 1.37
Rs: 0
Poroshell 120, 150mm Columns – HPLC or UHPLC
2 mL/min
min 0 2 4 6 8 10 12 14 16 18
mAU
0
20
40
60
80
100
120
min 0 5 10 15 20 25 30 35
mAU
0
20
40
60
80
100
120
1 mL/min P = 285 bar
P = 538 bar
Conditions: Column: Poroshell 120 EC-C18, 4.6 x 150mm, 2.7um Mobile Phase: Solvent A: Water with 0.1% Formic Acid
Solvent B: Acetonitrile 1200 SL controlled temperature at 25 °C 2 ul flow cell
1. Hydroquinone
2. Resourcinol
3. Catechol
4. Phenol
5. 4-Nitrophenol
6. p-cresol
7. o-cresol
8. 2-Nitrophenol
9. 3,4 di methyl phenol
10. 2,3 di methyl phenol
11. 2,5 di methyl phenol
12. 1-napthol
Gradient: 1mL/min
Time %B
6.0 5%
51 60%
Gradient: 2mL/min
Time %B
3.0 5%
25.5 60%
UHPLC/TOF (1290/6230) Can Identify More
Compounds in Less Time
1 .5
min
Time Composition
0.0 10% ACN
1.5 100% ACN
224 pesticides at 50 pg each
217 ionized & detected in positive
mode
(97%, Find by Formula)
2.1 x 50 mm x 1.8 micron
Eclipse Plus C-18
900 bar
1.5 mL/min
1290 Infinity
TOF fast acquisition rates (20Hz) ensure maximum throughput
Page 32
Ultrafast LC/MS Analysis for 15 Analyte Subset
1290 Infinity Applications
Peak Width 0.7 sec
RRHD Eclipse Plus C18
2.1x 50 mm, 1.8 um
750 bar
1 minute
Time Composition
0.0 10% ACN
1.5 100% ACN
Ultimate speed on a short column with ballistic gradient
Page 34
Injection Volume
Conversion
Detector Settings
recommendation
Gradient and
Isocratic Method
Conversion (auto-
detected)
Method Translator Makes Changes Easy
Let Agilent Method Translator Do the Math Basic Mode for Easy Transfer of Conventional Method to RRLC
Page 35
Original Method New Method
Detailed Output Detailed Input
Agilent Method Translator – Advanced Mode More Detaled Information, But Still Easy to Use
Page 36
http://www.chem.agilent.com/en-US/Products-Services/Instruments-
Systems/Liquid-Chromatography/pages/1200infinity_cost_calculator.aspx
“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
What Is Your Current Column Efficiency and
How Fast Do You Want To Run?
Page 38
Column
Length (mm)
Resolving
Power
N(5 µm)
Resolving
Power
N(3.5 µm)
Resolving
Power
N(1.8 µm)
Typical
Pressure
Bar (1.8 µm)
150 12,500 21,000 32,500 560
100 8,500 14,000 24,000 420
75 6000 10,500 17,000 320
50 4,200 7,000 12,000 210
30 N.A. 4,200 6,500 126
15 N.A. 2,100 2,500 55
Analysis Time
Peak Volume
Analysis
Time*
-33%
-50%
-67%
-80%
-90%
Solvent Usage
* Reduction in analysis time compared to 150 mm column
• pressure determined with 60:40 MeOH/water, 1ml/min, 4.6mm ID
First, Reduce Column Length
Page 39
5um 1.8um
Reduce column length by factor of 3
Quite often original method will have more resolution than is actually
needed and a reduction by 5 may be possible
3.5 um 1.8um
Reduce column length by factor of 2
Quite often original method will have more resolution than is actually
needed and a reduction by 3 is possible
Flow Modification - Different Diameter Columns
ml/minmm
mmml/min 21.0
4.6
2.11.0 i.e.
2
2 col.
2
column1
column21 col. Flow
.Diam
Diam.Flow
Page 40
Decreased Column Volume May Require
Conversion for Injection Volume
2 col.
column1
column21 col. Inj.Vol.
Volume
VolumeInj.Vol.
2 col.column1
column21 col. 4
2.0
4.020 i.e. μl
ml
mlμl
Page 41
Zorbax column volume = 3.14 x r2 x L x 0.6 (r and L in cm)
Reduce injection volume
Page 42
4.6 mm 3 mm
Reduction to allow for diameter change
2.1 mm 4.6 mm
= 0.4 x Original
= 0.2 x Original
x Reduction to allow for length change
150 mm 50 mm = 0.33 x Original
e.g. Original 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
Simple Isocratic Method Transfer for Speed
Step 1
• Reduce Column Length and Particle Size
• Maintain Flow Rate
Step 2 Faster
• Increase Flow Rate
• Pressure too High? Increase Temperature to Lower Pressure
• Increase Flow Rate
Step 3 Even Faster (Need Instrument Optimization)
• Decrease Column Diameter (2.1mm), Reduce Flow Rate Proportionately
• Reduce Injection Volume Based on Ratio of Column Volumes
• Increase Flow Rate
Page 43
Simplified Gradient Method Transfer
Step 1
• Reduce Column Length and Particle Size
• Adjust Gradient Time by same Factor
• Maintain Flow Rate
Step 2 Faster
• Increase Flow and Reduce Gradient Time
• Stop When Reach Flow Limits of Instrument
Step 3 Even Faster
• Decrease Diameter of Column (2.1mm)
• Match Flow to New Column Diameter
• Reduce Injection Volume
• Repeat Step 2 Until Reach 80-90 % Instrument Pressure Limit
Page 44
Group/Presentation Title
Agilent Restricted
Month ##, 200X
Let’s Look at Method Development of These
Compounds
diltiazem
dipyridamole
propranolol pindolol
disopyramide
β-blocker
Anti-arrhythmic
Vasodilator
Ca+ channel blocker
Group/Presentation Title
Agilent Restricted
Month ##, 200X
Start at Low pH, Adjust Organic
Eclipse Plus C18 Cardiac Drugs with Acetonitrile
2
mAU
0
100
200
300
400
2
mAU
0
25
50
75
100
125
min 2 4 6 8 10 12
mAU
-20
0
20
40
60
Column: ZORBAX RRHT Eclipse Plus C18, 4.6 x 50 mm, 1.8 mm
Mobile Phase: A: 25 mM NaH2PO4 , pH 3.0 B: ACN
Flow Rate: 2.0 mL/min Temperature: 30°C Detection: UV 240 nm
Sample: Cardiac Drugs 1.Pindolol 2. Diisopyridamide 3.Propranolol 4.Dipyridamole 5. Diltiazem
40% ACN
20% ACN
30% ACN
k= 44!!
RRHT Eclipse Plus C18 allows for very fast
optimization of % organic in mobile phase.
Good resolution
Fast analysis
No time wasted To get this k on a 25cm
column at 2 mL/min
would require 1.5 hours
run time!!
Group/Presentation Title
Agilent Restricted
Month ##, 200X
Start at Low pH on Eclipse Plus C18
Cardiac Drugs with Methanol, Changing Organic Modifier
2
mAU
0
100
200
300
400
2
mAU
0
25
50
75
100
125
min 2 4 6 8 10 12
mAU
0
20
40
60
80
Column: ZORBAX RRHT Eclipse Plus C18, 4.6 x 50 mm, 1.8 mm
Mobile Phase: A: 25 mM NaH2PO4 , pH 3.0 B: MeOH
Flow Rate: 2.0 mL/min Temperature: 30°C Detection: UV 240 nm
Sample: Cardiac Drugs 1.Pindolol 2. Diisopyridamide 3.Propranolol 4.Diltiazem 5. Dipyridamole
70% MeOH
40% MeOH
50% MeOH
k= 49!!
Adjusting MeOH influences
retention like adjusting ACN
Solvents can be compared
quickly with RRHT columns
MeOH changes selectivity and
gives longer analysis time for
these analytes
• When flow limit of pump is reached
Consider using a smaller i.d. column
• When approaching about 90% pressure limit of instrument
• 540 bar for 1200SL pump
• 360 bar for 1100/1200 pump
Remember to allow for increase in pressure at non-ideal mix
• When resolution is no longer satisfactory
When to Stop!?
Methanol/water 40C
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
0 20 40 60 80 100 120
%Methanol
Vis
co
sit
y
ACN/water 40C
0.00
0.20
0.40
0.60
0.80
0 20 40 60 80 100 120
%ACN
Vis
co
sit
y
Page 49
Agilent 1.8u and Poroshell 120 Columns
• Make High Resolution, Fast LC Possible on All Instruments
• Reduce Analysis Time
•Speed Up Method Development
•Improves Methods on HPLC
•Provides Best Benefits on UHPLC
•Coupled with UHPLC Instruments Allow Higher Flow Rates for
Higher Resolution Without Long Run Times
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