Transcript UHPLC Tips and Tricks

UHPLC Tips and Tricks:
Accelerate your Chromatography and
Enhance your Resolving Power
Agilent Technologies 2013
ASTS Houston, TX
Two Particle Technologies For
UHPLC Performance
SOLID
CORE
Sub 2um Totally
Porous Particle
Superficially Porous Particle
Why Use Sub-2um Particles?
Improved Efficiency over Wider Flow Rate Range
0.0030
HETP (cm)
0.0025
0.0020
5.0m
3.5m
1.8m
0.0015
0.0010
0.0005
0.0000
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Volumetric Flow Rate (mL/min)
Page
3 3
Page
4.0
4.5
5.0
Pressure Limit Under 1200bar?
Poroshell 120 columns have:
•
•
•
•
80-90% efficiency of sub 2um
At ~40-50% lower pressure
A 2.7um particle size
The particle has a solid core (1.7um) and
porous outer layer with a 0.5um diffusion
path
•
A 2um frit to reduce clogging
0.5um
1.7um
0.5um
4
Confidentiality Label
July 7, 2015
Typical Performance Comparisons of Various
Particles and Column Lengths
Length
N
5um
N
3.5um
N
1.8um
Bar
1.8um
N
Poro
120
Bar
Poro
120
150mm
12,500
21,000
32,500
560
~28,000
280
100mm
8,500
14,000
24,000
420
~20,000
210
50mm
4,200
7,000
12,000
210
~10,000
105
• pressure determined with 60:40 MeOH/water, 1ml/min, 4.6mm ID
Same Method and Very Similar Performance for Both Columns
Analysis of 15 Analgesic Compounds
3 x 100 mm, 1.8 µm
Ibuprofen:
PW1/2=0.012
3 x 100 mm, 2.7 µm
Ibuprofen:
PW1/2=0.012
2 min
June, 2011
Proportional Reduced Length/Particle Size
No Loss in Resolution
Columns: Eclipse Plus C18, as described below. Mobile Phase: A: water, B: MeOH, (15:85) Injection volume: 6uL
Temperature: 25°C Flow: 1 mL/min. Detection: 310, 4 nm, 0.5 s response time, semi-micro flow cell, Sample: Sunscreens
1
Rs3,2= 6.41
mAU
60
3
2
40
4.6 x 150 mm, 5 µm
P=82 bar
4
20
0
0
2
4
6
8
10
12
14
min
Rs3,2= 6.65
mAU
4.6 x 100 mm, 3.5 µm
P=105 bar
100
80
60
40
20
0
0
2
4
6
8
10
12
14
min
Rs3,2= 6.51
mAU
4.6 x 50 mm, 1.8 µm
P=208 bar
150
100
50
0
0
2
4
6
8
10
12
14
min
8330 Explosives Current Practice
First Screening Run- Column is C18 – Incomplete Resolution
mAU
25
7,10
EXPLOSIVES
20
3
1. HMX
6
1
15
(5ng/µL in ACN)
8,11
4
2
5
2. RDX
9
10
12 13
5
14
3. 1,3,5-Trinitrobenzene
4. 1,3-Dinitrobenzene
5. Nitrobenzene
0
6. 2,4,6-Trinitrotoluene
0
5
10
15
20
25
min
7. 2-Amino
mAU
-4,6-dinitrotoluene
25
8. 2,4-Dinitrotoluene
20
9. Tetryl
10. 4-Amino
15
-2,6-dinitrotoluene
3
10
1
4
5
2
0
0
9
5
5
6
10,8
11. 2,6-Dinitrotoluene
7
12. 2-Nitrotoluene
11
12 13
10
15
14
13. 4-Nitrotoluene
20
25
min
14. 3-Nitrotoluene
Sample: EPA8330 Explosives (5ng/µL each); Injection: (4µL);
Columns: Zorbax Eclipse XDB-C18, 4.6 x 100mm, 3.5µm, (P/N:961967-902 ); Hypersil BDS-C18 (4.0 x 150mm, 3µm)
Mobile Phase: A=H2O, B=Methanol; Gradient: 26–40%B in 10min, 40–55%B in 10min, 55–70%B in 10min, 70–26%B in
1min; Total=31min.
Flow rate: 0.72 mL/min; Temperature: 38°C; Detection: UV (Sig=235,40nm, Ref=360,100nm)
Page 8
Better Selectivity and More Efficient Column
Equal Fast, Complete Resolution
4.6 x 100mm, 1.8um Extend C18
1.312
D A D 1 B , S ig=250,60 R ef=360,100 (070803\E XTE N D _3X500115.D )
4.381
m AU
18.815
18.138
17.725
14.750
12.478
14.049
10
11.771
7.988
20
11.246
10.660
2.628
30
5.832
40
0
-10
0
2
4
6
8
10
12
14
16
18
m in
10
12
14
16
18
m in
P M P 1 , P ressure
MP: 75 A: 5mM NH4COOH (pH 6), 25 B: MeOH
At 15 min B=40%
Flow: 1.7 mL/min.
temp: 40C
2 uL injection x(50ug ea/mL)
bar
560
540
520
500
480
0
Page 9
2
4
6
8
Notoriously Difficult PAH 16 Component Test Mix
Includes Isomers of Different “Planarity” (Molecular Shape)
Acenaphthene
Benzo(b)flouranthene
Dibenz(a,h)anthracene
Naphthalene
Acenaphthylene
Benzo(k)flouranthene
Fluoranthene
Pyrene
Anthracene
Benzo(g,h,i)perylene
Fluorene
Benz(a)anthracene
Phenanthrene
Indeno(1,2,3-c,d)pyrene
Benzo(a)pyrene
Page 10
Chrysene
Conventional HPLC Assay for PAH 610 Mix
Eclipse PAH 4.6x 250mm, 5.0µm
mAU
1400
1200
1000
800
Rs = 2.2
600
400
1 = Toluene
2 = Naphthalene
3 = Acenaphthylene
4 = Acenaphthene
5 = Fluorene
6 = Phenanthrene
7 = Anthracene
8 = Fluoranthene
9 = Pyrene
10 = Benzo(a)anthracene
11 = Chrysene
12 = Benzo(b)fluoranthene
13 = Benzo(k)fluoeanthene
14 = Benzo(a)pyrene
15 = Dibenzo(a,h)anthracene
16 = Benzo(g,h,i)perylene
17 = indeno(1,2,3-c,d)pyrene
200
0
5
Page 11
10
15
20
25 min
PAH 610 Mix on 4.6x50mm, 1.8µm PAH Column
Much Faster and Resolution = 250mm, 5µm Column
mAU
2000
1750
1500
Rs = 2.2
1250
1000
750
500
250
0
1
Page 12
2
3
4
5
min
Resolution Equation for Gradient Elution
N
V
a k*
R
4
k* 
tg F
S (D%B)
D%B =
difference between initial and final
%B
S =
constant (≈ 4 for 100 - 500
Da)
F = flow rate (mL/min.)
Vm tg = gradient time (min.)
Vm = column void volume
(mL)
Zorbax Vm= π x (Col Internal Radius)2 x Length x 0.6
Page 13
Method Translator Makes Changes Easy
Let Agilent Method Translator Do the Math
Basic Mode for Easy Transfer of Conventional Method to RRLC
Injection Volume
Conversion
Detector Settings
recommendation
Gradient and
Isocratic Method
Conversion (autodetected)
Page 14
Conventional Column - 4.6 x 150mm, 5µm, SB-C18
Flow Rate
3.323
mAU
5.651
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\070809SBC180003.D)
Temperature 30°C
Wavelength 246nm
Sample rate
2.5 Hz
8.337
250
1.0 ml/min
6.114
200
10.982
100
9.690
5.064
6.964
150
1.474
50
0
0
2
4
6
8
10
12
Initial Pressure: 69 bar
Final Pressure: 38 bar
Page 15
14
16
Time (min)
% Acetonitrile
0
50
10
90
13.5
90
13.6
50
15
50
18
min
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
Flow Rate
140
Temperature
30°C
Wavelength 246nm
Sample rate
13.74 Hz
2.464
120
100
1.0 ml/min
3.357
1.176
mAU
2.254
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS 2007-08-09 17-25-25\070809SBC180009.D)
60
4.343
2.004
3.871
2.811
80
40
20
0
0
1
2
3
4
5
6
Initial Pressure: 132 bar
Final Pressure: 74 bar
Page 16
min
Time (min)
% Acetonitrile
0
3.33
4.5
4.53
5
50
90
90
50
50
Increase Column Flow-Reduce Gradient Time
Double Flow (2mL/min) – ½ Gradient Time
RRHT 4.6 x 50mm, 1.8µm, SB-C18
Flow Rate
0.587
1.146
mAU
1.715
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS 2007-08-10 08-22-16\070810SBC180004.D)
140
Temperature
30°C
Wavelength 246nm
Sample rate
13.74 Hz
1.260
120
100
2.0 ml/min
2.201
60
1.970
1.020
1.437
80
40
20
0
0
0.5
1
1.5
2
2.5
3
Initial Pressure: 266 bar
Final Pressure: 146 bar
Page 17
3.5
Time (min)
% Acetonitrile
0
1.67
2.25
2.27
50
90
90
50
4
3.34
4.5
min
50
Tips to Keep This From Happening!
Agilent Publication
Customer Results
18
Relationship of Column i.d. to
Flow Rate and Injection Volume
Standard Analytical Solvent Saver
Narrow Bore
Column
Cross Section
Column Internal
Diameter
4.6 mm
3.0 mm
2.1 mm
Flow Rate/min.
1.00 mL
0.40 mL
0.20 mL
Inj. Vol. Ratio
1
0.4
0.2
No Change in Flow as Column Diameter
Decreases-Reduced Resolution
R s = 1.65
R s = 1.32
R s = 0.95
4.6 x 150 mm
483 psi
3.0 x 150 mm
1135 psi
2.1 x 150 mm
2316 psi
0
1
2
3
4
5
6
Time (min)
Flow Rate: 1.0 mL/min
Confidentiality Label
20
July 7, 2015
Maintaining Linear Velocity on Columns of
Different i.d. Holds Resolution
4.6 x 150 mm
2.1 x 150 mm
3.0 x 150 mm
1
1
4
2
4
6
3
2
5
3
5
6
1
4
2
0
Time (min)
40
Flow Rate: 1.0 mL/min
Injected: 3 uL
Detector Cell Volume: 8 uL
0
Time (min)
40
Flow Rate: 0.5 mL/min
Injected: 2 uL
Detector Cell Volume: 8 uL
0
3
5
Time (min)
6
40
Flow Rate: 0.25 mL/min
Injected: 1 uL
Detector Cell Volume: 2 uL
Confidentiality Label
21
July 7, 2015
Instrument Impact on Column Performance
Gradient Delay or Dwell Volume
.
Extracolumn Volume
.
Number of
Scans or points
Data Sampling ( or Acquistion) Rate
Conversion for Fast and Ultra-Fast HPLC
1200 to 1260 Series LC Systems
Traditional LC Columns
Fast LC/UHPLC Columns
High pressure
Gradient pump
High pressure
Gradient pump
0.12 x 400 mm capillary
0.17 x 400 mm capillary
Std or Well
Plate sampler
Std or Well
Plate sampler
0.12 x 150 mm capillary
0.17 x 150 mm capillary
3 L
heat exchanger
0.17 x 105 mm capillary
Standard assembly
without standard mixer
3 L
heat exchanger
Thermostatted
Column
compartment
Thermostatted
Column
compartment
0.12 x 105 mm capillary
Rapid Resolution
HT Column
Rapid Resolution
HT Column
Diode Array
Detector
Diode Array
detector
0.12 x XX mm PEEK Capillary
Waste
* Pieces to upgrade, in kits
Mass
Spectrometer
Optimizing Connecting Tubing Volume
For UHPLC Columns
mAU
400
Resolution 0.961
System Tubing Volume Not Optimized
0.17mm i.d. tubing
Peak width 0.037 min
300
200
Peak width 0.038 min
100
0
0.5
1
1.5
2
mAU
600
550
2.5
min
Resolution 1.902
System Tubing Volume Optimized
0.12mm i.d. tubing
Peak width 0.019 min
400
350
200
Peak width 0.018 min
100
0
0.5
1
1.5
2
2.5
min
Effect of Data Acquisition Rate (time constant)
Peak Width, Resolution and Peak Capacity in Ultra-Fast LC
80Hz vvsvs.us 20Hz
– 30% Peak Width
+30% Resolution
+ 40% Peak Capacity
+ 70% Apparent Column Efficiency
PW=0.30sec
80Hz
PW = 0.33 sec
40Hz
PW=0.42sec
80Hz vervs.s 10Hz
– 55% Peak Width
+ 90% Resolution
•
+ 120% Peak Capacity
+ 260% Apparent Column Efficiency
20 Hz
PW=0.67sec
10Hz
PW=1.24sec
0
0.1
0.2
0.3
0.4
5Hz
0.5
min
Max Performance RRHD - UHPLC/TOF (1290/6230)
Identify More Compounds in Very Short Run Time
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
1 .5
min
Time
Page 26
Composition
0.0
10% ACN
1.5
100% ACN
Very Narrow Peaks in MS Require More
Scans/Second –Optimize Scan Speed!
Peak Width 0.7 sec
1290 Infinity Applications
What Happens If the Connections Poorly Made ?
Wrong … too long
Ferrule cannot seat properly
X
Wrong … too
short
Mixing Chamber
If Dimension X is too long, leaks will occur
X
If Dimension X is too short, a dead-volume,
or mixing chamber, will occur
Page 28
Influence of Bad Post-Column Connection
mAU
One bad capillary connection!
130 mAU
140
120
100
80
60
40
20
0
0
0.1
0.2
0.3
0.4
min
mAU
210
180
150
120
90
60
30
0
Fixed!
160 mAU
0
Page 29
0.1
0.2
0.3
0.4
min
Summary
• Two Particle Technologies – Sub-2 and Superficially Porous
• 2x-3X speed increase needs little more than a column change
• Same I.D., shorter Column, smaller Particle
• 5x+ Speed or Sensitivity May Require a 2.1mm I.D. Column
• 2.1mm will require optimizing extra-column volume in most
instruments
• Kits are available to improve instrument performance
• Maximum performance will be realized on new design LCs
• Very complex samples will need UHPLC instruments and columns
APPENDIX
A Few Extra Slides to Illustrate Points of Interest
Comparison of 4.6 x 250 mm 5 um to Poroshell 120
EC-C18 4.6 x 100 mm, 2.7um
110 bar
40
23.076
29.290
16.151
16.435
15.248
12.674
11.596
20.687
60
11.116
80
9.712
mAU
100
Mobile Phase:
A: 0.1% formic acid in Water
B: 0.1% formic acid in ACN
Time
%B
0 8
33 33
34 33
Column: Eclipse Plus C18
4.6 x 250mm, 5um
Flow Rate: 1 mL/min
Sulfadiazine,
Sulfathiazole
Sulfapyridine
Sulfamerazine,
Sulfamethazine,
Sulfamethazole,
Sulfamethoxypyridazine,
Sulfachloropyridazine
Sulfamethoxazole,
Sulfadimethoxine
20
0
100
15
20
5.450
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
5.920
4.4374.558
2.606
1.719
10
3.867
150
2.189
200
2.311
mAU
250
7.037
5
25
30
min
25
30
min
325 bar
50
0
5
10
15
20
11.596
Expand High Speed Chromatograms for True
Comparison to Slower Separation
29.290
110 bar
23.076
20.687
15.248
9.712
60
11.116
80
16.151
16.435
12.674
mAU
100
40
20
100
25
30
min
5.450
5.920
2.606
2.189
1.719
200
150
20
4.558
325 bar
15
4.437
250
10
3.867
5
2.311
mAU
7.037
0
50
0
5
min
8330 Explosives Current Practice
2nd Confirmation Run on Orthogonal Phase (CN) – Incomplete Resolution
Lack of complete resolution doubles work for positive screening
DAD1 A, Sig=214,10 Ref =360,100 (EPACN\EXPSTD02.D)
UV=214 nm
mAU
3,4
25
20
(5ng/µL in ACN)
10
2. RDX
6
2
5
5
1. HMX
7,10
13,12 11
8
15
EXPLOSIVES
1
9
3. 1,3,5-Trinitrobenzene
4. 1,3-Dinitrobenzene
14
5. Nitrobenzene
0
6. 2,4,6-Trinitrotoluene
0
5
10
DAD1 B, Sig=235,40 Ref =360,100 (EPACN\EXPSTD02.D)
15
20
min
7. 2-Amino
-4,6-dinitrotoluene
UV=235 nm
3,4
mAU
25
8. 2,4-Dinitrotoluene
25
9. Tetryl
20
15
13,12
10
5
5
10. 4-Amino
7,10
8 11
-2,6-dinitrotoluene
6
2
1
11. 2,6-Dinitrotoluene
9
12. 2-Nitrotoluene
14
13. 4-Nitrotoluene
0
14. 3-Nitrotoluene
0
5
10
15
20
25
min
Sample: EPA-8330 Explosives (5ng/µL each); Injection: (4µL); Column: Zorbax Eclipse XDB-CN, 4.6 x 100mm, 3.5µm, (P/N:961967-905 )
Mobile Phase: A=H2O, B=Methanol; Gradient: 26–40%B in 10min, 40–55%B in 10min, 55–70%B in 10min, 70–26%B in 1min; Total=31min.
Flow rate: 0.72 mL/min; Temperature: 38°C; Detection: UV (Sig=214,10nm, Ref=360,100nm; Sig=235,40nm, Ref=360,100nm)
Page 34
Small Particles Are Rugged and Robust
Overlay of Runs # 2, 500, 900 injections
1 = Toluene
2 = Naphthalene
3 = Acenaphthylene
4 = Acenaphthene
5 = Fluorene
6 = Phenanthrene
7 = Anthracene
8 = Fluoranthene
9 = Pyrene
10 = Benzo(a)anthracene
11 = Chrysene
12 = Benzo(b)fluoranthene
13 = Benzo(k)fluoeanthene
14 = Benzo(a)pyrene
15 = Dibenzo(a,h)anthracene
16 = Benzo(g,h,i)perylene
17 = indeno(1,2,3-c,d)pyrene
mAU
350
300
250
200
150
100
50
0
0
Page 35
1
2
3
4
5
6
min