3 x 100 mm Poroshell 120 Columns

Optimal
Design
For
A
Mass
Spectrometer
Vacuum-pump
Cover
that
Optimization of HPLC Instrumentation for Use with Poroshell 120 Columns
Poster Number 2080-9
Reduces
Pump
Noise
and
Facilitates
Oil
Changes
William J. Long, Anne E. Mack, John W. Henderson Jr. Agilent Technologies 2850 Centerville Rd. Wilmington Del. USA 19808
Pittcon 2010
able to deliver similar performance to sub-two micron materials
at
substantially
lower
pressures.
While
modern
Optimize detector settings by adjusting the scan rate and/or the
time constant to the fastest possible settings such that signal-tonoise (S/N) is not adversely affected.
HPLC
instrumentation and data systems are able to capture the
benefits of these particles, attention to the instrumentation
The Peakwidth control in ChemStation enables you to select the
peak width(response time) for your analysis. The peak width (as
defined in the ChemStation software) is the width of a peak at
half height. Set the peak width to the narrowest expected peak
in your sample. With Poroshell 120 column expect narrow
peaks, similar to those generated with sub 2 micron columns.
Set the detector to the fastest setting, then to the second fastest
setting and evaluate if the signal to noise levels are different.
configuration is important if optimal results are desired.
Configuration should be optimized with regard to the speed
with which peaks pass through the detector and the small peak
volumes produced in the system. Peak volumes of 50 to 75 %
lower volume as compared to conventional 5 and 3.5 micron
6 mm flow cell
3mm flow cell
15000
k=0.5
k=1.6
10000
Inside Diameter (mm) Length (mm) Material Color
0.12
180
SS
Red
0.12
280
SS
Red
0.12
105
SS
Red
0.12
150
SS
Red
0.12
105
SS
Red
0.12
150
SS
Red
0.12
280
SS
Red
0.12
400
SS
Red
20000
15000
k=0.5
k=1.6
10000
k=3
k=3
5000
5000
Overlay of Van Deemter Plots
25000
20000
20000
Optimal Flow Rate for Poroshell Faster than for 5
or 3.5 micron Columns it Replaces
Use of 0.12 mm Tubing instead of 0.17 mm Tubing
This reduces extra column volume by half.
10 mm flow cell
25000
25000
Optimize Flow Rate
Connections
15000
k=0.5
0.17
0.17
0.17
0.17
0.17
0.17
0.17
0.17
k=1.6
10000
k=3
5000
Column part number
695975-302
180
280
130
90
105
150
280
400
SS
SS
SS
SS
SS
SS
SS
SS
Connections
1 end pre-swaged
1 end pre-swaged
1 end pre-swaged
pre-swaged
Without fittings
Without fittings
Without fittings
Without fittings
Green
Green
Green
Green
Green
Green
Green
Green
1 end pre-swaged
1 end pre-swaged
1 end pre-swaged
1 end pre-swaged
Without fittings
Without fittings
Without fittings
Without fittings
12
Part Number Volume (ul)
G1313-87304
2.0
01090-87610
3.2
01090-87611
1.2
G1315-87312
1.7
5021-1820
1.2
5021-1821
1.7
5021-1822
3.2
5021-1823
4.5
G1313-87305
01090-87304
01090-87305
G1316-87300
5021-1816
5021-1817
5021-1818
5021-1819
10
USE LOWER VOLUME
RED TUBING WHEN
POSSIBLE
Probe:Heptanophenone
Solvent 60/40 MeCN/Water
Temperature 25 C
8
Agilent Poroshell 120 EC-C18, 4.6 mm x 50 mm, 2.7 um
(CFU-1161)
4.1
6.4
2.9
2.0
2.4
3.4
6.4
9.1
6
Agilent ZORBAX Eclipse Plus C18, 4.6 mm x 50 mm, 1.8 um
(USUXG04047)
Agilent ZORBAX Eclipse Plus C18, 4.6 mm x 50 mm, 3.5 um
(USBH006534)
4
GREEN TUBING HAS
2 x VOLUME OF RED
TUBING FOR EQUAL
LENGTH
Agilent ZORBAX Eclipse Plus C18, 4.6 mm x 50 mm, 5.0 um
(USFA002925)
2
Column Flow Rate for Suggested
Recommended
Analysis with Poroshell Columns :
Using
a
4.6
mm
column
2
ml/min
Diameter
Starting Flow Rate
Using a 3 mm column 0.85 ml/min
Using a 2.1 mm column 0.42 for
ml/minOptimization
0
0
2
4
6
8
10
12
u (mm/s)
particle filled columns of similar size are found. The most
relevant system parameters are detector response time,
Larger Flow Cells, used at Fast Data Collection speeds can
be used with Larger Columns as shown with this
3 x100 mm Poroshell 120 EC-C18 Column
Orlando FL,USA
h
Optimize Detector Speed
Superficially porous HPLC columns, such as Poroshell 120 are
Efficiency (N)
Optimization of
Efficiency (N)
Introduction
Robert D. Ricker, Bernard J. Permar II, Lindy Miller, Jerome Szczepaniak, Viet Nguyen, and Patricia Castelli
Agilent Technologies, 2850 Centerville Rd., Life Science/Chemical Analysis, Wilmington, DE 19808, USA
Data Collection Rate Combined with
Data Collection
Proper
Column
Flow Cell Choice
Efficiency (N)
SA
Pittcon 2006
Orlando, FL, USA
0
0
0
50
100
0
Data Collection Rate (Hertz)
Data Collection Rate Importance
100
0
Data Collection Rate (Hertz)
Flow Cells
Effect of Data Collection Rate using a 2.1 x 100 mm Poroshell 120 EC-C18
25000
of these parameters is optimized and de-optimized. The
Path Length
50
100
Data Collection Rate (Hertz)
Volume
Part Number
Standard (with RFID tag)
10 mm
13 µl
G1315-60022
Semi-micro (with RFID tag)
6 mm
5 µl
G1315-60025
Micro (with RFID tag)
20000
combined effect of using all three in an optimized and a de-
50
2 ul QC Mix, Uracil, Phenol (k=0.5), 4-Chloronitrobenzene(k=2), Napthalene(k=3.8) 60% MeCN 40 % Water 0.8 ml/min micro flow cell
connecting tubing volume and flow cell volume. Comparisons
of chromatographic performance will be shown in which each
1>k’>10
0
3 mm
2 µl
Peak tailing/fronting
Peak Capacity of Poroshell 120 is slightly
lower than RRHT but at a lower pressure
What Happens If the Connections Poorly Made ?
Wrong … too long
G1315-60024
4.6 mm
2 ml/min
3.0 mm
0.85 ml/min
2.1 mm
0.42 ml/mi
DAD1 A, Sig=270,8 Ref=off (PHENOLS\PHENOLS 2010-01-20 10-48-05\ECC18_2000004.D)
mAU
Average Peak Capacity
20
391 bar 3.5 ml/min
0
70.0
Ferrule cannot seat properly
5 min
40
Pressure >600 Bar
0
5
DAD1 A, Sig=270,8 Ref=off (PHENOLS\PHENOLS 2010-01-19 16-24-38\ECC18000003.D)
10
15
20
mAU
25
min
329 bar 3.0 ml/min
40
20
0
5
DAD1 A, Sig=270,8 Ref=off (PHENOLS\PHENOLS 2010-01-19 16-24-38\ECC18000006.D)
k=0.5
k=2
10000
k=3.8
Optimize the Whole Experiment
0
Minimize Extra Column Volume
Wrong … too short
X
Mixing Chamber
Instrument Contributes to Column Performance
Poroshell 120 EC C18
Competitor C18 2.5 um
30.0
Eclipse Plus C18 5 um
Eclipse Plus C18 1.8 um
20.0
40
50
60
70
80
10
15
20
90
min
25
min
220 bar 2.0 ml/min
40
20
0
0
5
DAD1 A, Sig=270,8 Ref=off (PHENOLS\PHENOLS 2010-01-19 16-24-38\ECC18000014.D)
10
15
20
mAU
25
20
0
0
5
DAD1 A, Sig=270,8 Ref=off (PHENOLS\PHENOLS 2010-01-19 16-24-38\ECC18000018.D)
10
15
20
25
min
mAU
111 bar 1.0 ml/min
40
0
Using Agilent 1200SL w/80 Hz Detection
10.0
min
166 bar 1.5 ml/min
40
20
0
30
25
mAU
40.0
Eclipse Plus 3.5 um
20
20
274 bar 2.5 ml/min
0
5
DAD1 A, Sig=270,8 Ref=off (PHENOLS\PHENOLS 2010-01-19 16-24-38\ECC18000010.D)
If Dimension X is too long, leaks will occur
10
15
40
20
0
50.0
5000
0
10
mAU
Peak Capacity
given to help the chromatographer achieve best results.
Efficiency (N)
optimized mode will emphasize the results. Guidelines will be
60.0
15000
0
5
DAD1 A, Sig=270,8 Ref=off (PHENOLS\PHENOLS 2010-01-19 16-24-38\ECC18000022.D)
10
15
20
25
min
mAU
56 bar 0.5 ml/min
40
Data Collection Rate (Hz)
1. Optimize the data collection rate of the detector. Set the data
collection rate to the fastest setting such that signal to noise (S/N)
is not adversely affected.
2. Choose the flow cell carefully. The standard flow cell on the
Agilent 1200 has a volume of 13 µl. This may diminish the
performance achievable using Poroshell 120 columns. Smaller
volume flow cells such as the semi micro (6 mm/5 µl)or micro (3
mm/2 µl) are recommended for best performance.
3. Minimize Extra Column Volume. Use the shortest length of tubing
possible for all connections. Use Red (0.12 mm id) tubing instead
of Green (0.17 mm) as it has only half of the volume of the larger
green tubing. Use proper fittings for all connections. Make zero
dead volume connections. These will ensure best peak shape.
Chromatography Optimization
1. Optimize the flow rate to achieve the desired efficiency (N)
or peak capacity (Pc).
Column part number 695775-902
0
1 ul QC Mix, Uracil, Phenol (k=0.5), 4-Chloronitrobenzene(k=2), Napthalene(k=3.8) 55% MeCN 45 % Water 0.55 ml/min micro flow cell
Slower data collection is practical when analytes retain longer. (k’> 4)
Mobile
Phase
Choice of Flow Cell
Data
Device
Flow Cells are an integral part of HPLC instrumentation. The
tendency is to use a larger flow cell in order to more easily
detect the compounds of interest is common. However, as
shown in this work, they can also be a source of extra column
volume. Choose the flow cell carefully.
While detector speed can compensate for excessive flow cell
dispersion, an appropriate flow cell should be used,
The volume of a Standard flow cells for an Agilent 1100 or 1200
system is 10 µL.
20
10
6
k=0.5
k=2
10000
600
40
500
30
400
10
min
0
0
2
4
6
8
10
min
0
2
4
DAD1 A, Sig=254,4 Ref=360,100 (MERCURY 20...AND PRESS LOAD ECLIPSE PLUS C18 3X100, 1.8\USUYB0145500037.D)
mAU
500
400
15000
8
10
min
Cause:
Capillary tubing connecting ALS and column was swaged improperly on
the ALS end (ferrule was flush with end of tubing, causing a void).
Solution:
Replace tubing (bottom chromatogram).
600
20000
6
300
200
k=0.5
100
k=2
10000
0
k=3.9
2
4
6
8
10
min
5000
1
2
3
4
5
6
7
Column part number 695775-902
1 ul QC Mix, Uracil, Phenol (k=0.5), 4-Chloronitrobenzene(k=2), Napthalene(k=3.8) 55% MeCN 45 % Water 0.55 ml/min micro flow cell
3 x 100 mm Poroshell 120 Columns
(early eluting compounds are less effected by extra column volume than with
2.1 x 100 mm Poroshell 120 columns)
Which type is used and when?
Stainless Steel (SS) fittings are the best choice for reliable high
pressure sealing
Agilent uses Swagelok type fittings with front and back
ferrules, which give best sealing performance
throughout our LC system (use this on the instrument
connections, i.e. valves, heaters etc)
PEEK (<400 bar system pressure) fittings are ideal where:
20000
k=0.5
15000
k=1.6
10000
8
10
Flow Cell Path Length (mm)
12
Connections are changed frequently, i.e. connecting
columns
Bio-compatibility is needed
Pressure is less critical
Use this fitting on column connections with Poroshell 120
(PN 5042-8957)
Some typical column connectors shown here
0
Column part number 695775-902
1 ul QC Mix, Uracil, Phenol (k=0.5), 4-Chloronitrobenzene(k=2), Napthalene (k=3.8) 55% MeCN 45 % Water 0.55 ml/min micro flow cell
2. Excellent results can be achieved achieved on Agilent 1200 RRLC
or 1290 Infinity
1.
2.
3.
4.
5.
Optimize data collection rate (40 Hz detector with fast response time)
Use the smallest flow cell that you have ( 3mm micro flow cell works best
when using small columns).
Minimize extra column volume: flow cell and connecting tubing are the
biggest contributors, but needle seats should be replaced with smaller
volume seats. Red tubing has half the volume of green tubing.
Take care to make proper connections.
Optimize flow rates for best performance. Start with flow rate 2 ml/min on a
4.6 mm column, 0.85 ml/min on a 3.0 or 0.42 ml/min on 2.1 mm column.
Helpful References
1. Agilent 1200 Series Diode Array and Multiple Wavelength Detector
SL User Manual. G1315-90011, February 2006.
2. Maintaining Your Agilent LC and LC/MS Systems, 5990-4957EN,
November 2009.
3. “The Influence of Sub-Two Micron Particles on HPLC Performance”
5988-9251EN.
4. “Step-by-step upgrade of Agilent 1100 Series LC systems to Agilent
1200 Series Rapid Resolution LC systems for higher performance”,
Part 1. 5989-6336EN.
k=3
0
6
25 min
8
5000
4
Solvent A: Water with 0.1 % Formic Acid
Solvent B: Acetonitrile Scaled Gradients
1200 SL controlled temperature at 25 C
2 mm flow cell
Stainless Steel and Polymer Fittings
Polyketone fittings can be used up to 600 bar
2
4
10
100
5000
0
3.5
min
3. In order to achieve best performance
20
200
25000
8
After troubleshooting
mAU
Extra Column Effects on 2.1 x 100 mm Poroshell 120 Columns
Efficiency (N)
Efficiency (N)
15000
3
25
1. Poroshell 120 columns can achieve similar efficiencies as sub 2
micron columns with substantially less pressure.
30
0
2
4
DAD1 A, Sig=254,4 Ref=360,100 (MERCURY 20...AND PRESS LOAD ECLIPSE PLUS C18 3X100, 1.8\USUYB0145500037.D)
25000
30 % loss of efficiency with
a 10 mm standard flow cell
With 2.1 mm columns, it is
best to use a 3 mm flow
cell.
2.5
20
Conclusion
0
Extra Column Effects on 3 x 100 mm Poroshell 120 Columns
k=3.8
It included a G1312 B Pump, a G1367A Autosampler and a G1315CDAD SL
Detector. Data was collected and processed using ChemStation version B
3.01
Problem:
All peaks tail
(top chromatogram).
300
25000
20000
2
15
40
2.1 x 100 mm Poroshell 120 Columns (early eluters are
more severely affected by extra column broadening)
0
Flow Cell Choices
1.5
10
DAD1 A, Sig=254,4 Ref=360,100 (MERCURY 20... DATA; MERCURY 2010 ECLIPSE PLUS 3X100,1.8\USUYB0145300037.D)
0
Flow Cell Choices with a 2.1 x100 mm Poroshell
120 EC-C18
1
5
mAU
Extra Column Volume = sample volume + connecting
tube volume + fitting volume + detector cell volume
For best results, replace standard flow cells with 5 µL flow cells
(2 µL when using 2.1 mm ID columns)
0.5
Flow Rate (ml/min) scaled gradient
LC troubleshooting: poor peak shape, improperly
Before troubleshooting
swaged tubing
DAD1 A, Sig=254,4 Ref=360,100 (MERCURY 20... DATA; MERCURY 2010 ECLIPSE PLUS 3X100,1.8\USUYB0145300037.D) mAU
1. Use an injection solvent that is weaker than the mobile phase,
especially when using an isocratic method.
This work was performed on an Agilent 1200 RRLC
0
For best results when using small columns, collect data at 40 Hz.
3. Minimize sample dispersion in the column.
2. Gradients can minimize dispersion but be aware of possible effects.
0.0
If Dimension X is too short, a deadvolume, or mixing chamber, will
occur
Tubing Volume (ul)
2. If adapting an older method to a new Poroshell 120 column
properly scale the gradient and injection volume to the new
smaller column to quickly transfer the method and avoid
overloading.
0
X
Efficiency (N)
System Optimization
20
0
1
2
3
4
5
Tubing Volume (ul)
6
7
8
5. “Step-by-step upgrade of Agilent 1100 Series LC systems to Agilent
1200 Series Rapid Resolution LC systems for higher performance”,
Part 2. 5989-6337EN.
6. “Optimize Data Sampling Rate to Take Advantage of RRHT
Columns”, 5989-5810EN.
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