agilent poroshell 120 hplc columns- boost your lc productivity · agilent poroshell 120 hplc...
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5/3/2010
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Agilent Poroshell 120HPLC Columns-
Boost your LC Productivity
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Poroshell 120 – A New Approach to High Resolution and High Speed LC• Poroshell 120 is a high efficiency, high resolution column choice for enhancing productivity on an HPLC or UHPLC.
• Use by 1200, 1100 and other HPLC users (P < 400 bar) for fast separations with high resolution.
> 4.6 and 3.0 mm ID columns have higher column volumes for any HPLC
> 2.1 mm ID columns are good for LC/MS and on systems optimized for low column volume use
• Use by 1200 RRLC, 1290 Infinity LC or other UHPLC users with P > 400 bar
> Long Poroshell 120 columns deliver very high efficiency (single or series)
May 3, 2010
Confidentiality LabelPage 2
5/3/2010
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Poroshell 120 – A New Approach to High Resolution and High Speed LC• Poroshell 120 is a high efficiency, high resolution column choice for enhancing productivity on an HPLC or UHPLC.
• Use by 1200, 1100 and other HPLC users (P < 400 bar) for fast separations with high resolution.
> 4.6 and 3.0 mm ID columns have higher column volumes for any HPLC
> 2.1 mm ID columns are good for LC/MS and on systems optimized for low column volume use
• Use by 1200 RRLC, 1290 Infinity LC or other UHPLC users with P > 400 bar
> Long Poroshell 120 columns deliver very high efficiency (single or series)
May 3, 2010
Confidentiality LabelPage 3
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 for HPLC or
UHPLC use
• The particle has a solid core (1.7um) and porous outer layer with a 0.5um diffusion path
May 3, 2010Confidentiality Label
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Poroshell 120 columns have:
1.7um
0.5um
0.5um
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Poroshell 120 Columns Deliver
High speed, high resolution separations at HPLC pressures
> Compatibility with LC instruments – even with 150mm long columns!
> High efficiency columns mean high resolution and ability to run at high flow rates
High resolution separations at UHPLC pressures
An additional solution from Agilent for greater productivity
> Only Agilent has superficially porous 2.7um and totally porous sub 2um
> Solutions to meet customer needs – not an explanation of why the one product the competition has will meet every need
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Poroshell 120 is Efficient!
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Flow Rate: 0.582 mL/minAcetonitrile/Water (60:40)Injection Volume: 4 µL TCC: 26 oCDAD: Sig=254,4nm Ref=360,100nm
Sample: RRLC Checkout Sample (PN 5188-6529) spiked w/ 50 µL 2 mg/mL Thiourea in water/acetonitrile (65:35)
2.5 5 7.5 10
mAU
0
200
400
600
0.64
60.
826
1.14
8
1.51
8
2.01
62.
223
2.81
6
4.13
2
6.27
3
9.75
3
← N = 25053, Press = 182 bar
Poroshell 120 EC-C18, 3.0 x 100 mm, 2.7 um
min2.5 5 7.5 10 12.5
mAU
0
200
400
600
0.64
90.
865
1.27
5
1.75
6
2.41
82.
705
3.49
4
5.28
1
8.21
8
13.0
41
← N = 27295, Press = 386 bar
Eclipse Plus C18, 3.0 x 100 mm, 1.8 um
>90% of the efficiency of 1.8um
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High Efficiency on Poroshell 120 EC-C18 with Low Pressure
Column: Poroshell 120 EC-C18, 4.6 x 100mm PN 695975-902 Instrument: 1200 SL 2ul flow cell Flow Rate: 2.00 ml/min Sample: 2ul injection of 3B Mobile Phase: 60:40 MeCN:Water
min0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25
25,705
25,080
P= 254 bar
QC TestMeasure efficiency with neutral compounds
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High Efficiency at Low Pressure with Poroshell 120
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Column: Poroshell 120 EC-C18, 3.0 x 100mm, 2.7um
Flow Rate: 0.582 mL/minPressure: 184 barAcetonitrile/Water (60:40)Injection Volume: 4 µLTCC: 26 oCDAD: Sig=254,4nm Ref=360,100nmSample: RRLC Checkout Sample (PN 5188-6529), alkylphenones spiked w/ 50 µL 2 mg/mL Thiourea in water/acetonitrile (65:35)
min2.5 5 7.5 10
mAU
0
100
200
300
400
500
600
0.6
46
0.8
26
1.1
48
1.5
18
2.0
16
2.2
23
2.8
16
4.1
32
6.2
73
9.7
53
N = 25053
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Choose Poroshell 120 for High Efficiency HPLC
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min1 2 3 4 5 6
mAU
0
100
200
300
min1 2 3 4 5 6 7
mAU
0
100
200
300
min1 2 3 4 5 6 7
mAU
050
100150200250
Poroshell 120 EC-C18, 3.0 x 100mm, 2.7 µm; N = 23,283, P = 314 bar
RRHT Eclipse Plus C18 3.0 x 100mm, 1.8 µm;N = 25,364, P = 456 bar
Rapid Resolution Eclipse Plus C18, 3.0 x 100mm 3.5 µm;N = 11896, P = 153 bar
Columns: 3.0 x 100mm, Mobile Phase: 65% A: 0.2% F ormic Acid: 35% B: Methanol Isocratic, Flow Rate 0.5 mL/min 1 µL injection 26 °C Sig = 220, 4 nm, Ref = Off Sample: 1. Sac charin 2. Caffeine 3. P-hydroxybenzoic acid 4. Aspartame 5. Dehydroacetic acid 6. Benzoic acid
>90% Efficiency of sub 2um2X efficiency of 3.5umHPLC Pressure (< 400 bar)
2 4 6
mAU
0
100
200
300
2 4
mAU
0
100
200
300
min2 4
mAU
0
100
200
Poroshell 120 EC-C18 for Fast UHPLC Separations
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Column: Poroshell 120 EC-C18 3.0 x 100mm, 2.7um Mobile Phase: 65% A: 0.2% Formic Acid: 35% B: Methanol Isocratic, Flow Rate varies 1 µL injection 26 oC Sig = 220, 4 nm, Ref = Off Sample: 1. Saccharin
2. Caffeine 3. P-hydroxybenzoic acid 4. Aspartame 5. Dehydroacetic acid 6. Benzoic acid
Flow Rate = 0.5 mL/min, P = 300 bar, NBA = 24567
Flow Rate = 0.75 mL/min, P = 433 bar
Flow Rate = 1.0 mL/min, P = 559 bar
3
More viscous solvents like methanol can be used at HPLC or UHPLC pressures.
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Who will love to use Poroshell 120
HPLC users – needing greater productivity but without a UHPLC instrument or concerned with transferring methods to those without a UHPLC
> Pharma development before pharma discovery, more likely to have HPLC or need to confirm method on HPLC
> Leads to easy transition to QA/QC
> Generic pharma – less likely to have UHPLC
> USP methods – can substitute for 5um methods (-50% on particle size) for improved resolution
Page 11
Who will also love to use Poroshell 120
Dirty samples
> Pharma – DMPK early or late
> Clinical – many dirty samples, have been unwilling to go to sub 2um
> Food safety/environmental – extracts of food/soil considered dirty
> Customers who use Quechers with LC – samples not quite as clean as SPE
UHPLC users
> Primary goal is more resolution
> Product currently limited to 600 bar
> May want 4.6 mm ID or have a UHPLC in place of an LC and still prefer lower pressure separations
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Complete List of C18 Part Numbers (March 15 th)Poroshell 120 EC-C18, 2.7um
Part Number Poroshell 120 SB-C18, 2.7um
Part Number
4.6 x 150mm 693975-902 4.6 x 150mm 683975-902
4.6 x 100mm 695975-902 4.6 x 100mm 685975-9024.6 x 75mm 697975-902 4.6 x 75mm 687975-902
4.6 x 50mm 699975-902 4.6 x 50mm 689975-9024.6 x 30mm 691975-902 4.6 x 30mm 681975-9023.0 x 150mm 693975-302 3.0 x 150mm 683975-3023.0 x 100mm 695975-302 3.0 x 100mm 685975-3023.0 x 75mm 697975-302 3.0 x 75mm 687975-9023.0 x 50mm 699975-302 3.0 x 50mm 689975-302
3.0 x 30mm 691975-302 3.0 x 30mm 681975-302
2.1 x 150mm 693775-902 2.1 x 150mm 683775-902
2.1 x 100mm 695775-902 2.1 x 100mm 685775-902
2.1 x 75mm 697775-902 2.1 x 75mm 687775-902
2.1 x 50mm 699775-902 2.1 x 50mm 689775-9022.1 x 30mm 691775-902 2.1 x 30mm 681775-902
Available Now
Available March 15
How Do You Use Poroshell 120 Columns
• For HPLC and UHPLC!
• Do you need special instruments? NO
• Are there special sample precautions to take with dirty samples? NO
• For USP Methods? YES
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What Types of Instruments Can You Use Poroshell 120 Columns on? HPLC’s and UHPLC’sUHPLC considerations
> Poroshell 120 columns have a 600 bar pressure limit
> UHPLC is likely set up for low volume columns so columns can be used on all UHPLC configured systems
HPLC considerations
> Poroshell 120 columns work well below 400 bar
> Minimize extra column volume for the 2.1 and 3.0mm ID columns – follow instructions given in 2006 for RRHT columns
> Use adequate detector speed for sharp, efficiency peaks
Page 15
Comparison of Peak Efficiency on Poroshell 120 EC-C18 with Different Data Collection Rates
Column: Poroshell 120 EC-C18, 4.6 x 100mm Instrument: 1200 SL 2ul flow cell Flow Rate: 2.00 ml/min Sample: 2ul injection of 3B Mobile Phase: 60:40 MeCN:Water
min0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25
mAU
0
10
20
min0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25
mAU
0
10
20
min0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25
mAU
05
1015
min0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25
mAU
05
1015 10 Hz
20 Hz
40 Hz
80 Hz
23,87221,712
24,699
25,705
25,706 25,061
25,080
24,506
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Comparison of Peak Efficiency on Poroshell 120 EC-C18 with Different Data Collection Rates
Response Time
Hz Uracil Phenol 4-Chloro-Nitrobenzene
Naphthalene
Peak Width0.008
Peak Width0.011
Peak Width 0.021
Peak Width 0.031
0.02 80 16,240 21,641 25,706 25,061
0.1 40 15,414 22,028 25,705 25,080
0.2 20 12,755 18,647 24,699 24,506
0.5 10 6,965 12,658 21,712 23,872
Column: Poroshell 120 EC-C18, 4.6 x 100mm Instrument: 1200 SL 2ul flow cell Flow Rate: 2.00 ml/min Sample: 2ul injection of 3B Mobile Phase: 60:40 MeCN:Water
The 40Hz data rate is ideal based on the narrow peak widths obtained with the Poroshell 120 column.
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Separation of 12 Phenols on Poroshell 120 EC-C18 in 5 minutes – under 400 bar
274 bar 2.5 ml/min
min0 5
Conditions: Column: Poroshell 120 EC-C18, 4.6 x 50mm, 2.7umMobile Phase:Solvent A: Water with 0.1% Formic AcidSolvent B: AcetonitrileGradient::Time %B0.8 5%6.8 60%1200 SL controlled temperature at 25 C2 mm flow cell
1. Hydroquinone2. Resourcinol3. Catechol4. Phenol5. 4-Nitrophenol6. p-cresol7. o-cresol8. 2-Nitrophenol9. 3,4 di methyl phenol10. 2,3 di methyl phenol11. 2,5 di methyl phenol12. 1-napthol
Poroshell 120 gives high efficiency, high resolutio n separations quickly at HPLC pressures.
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Improve Resolution of 12 Phenols on Poroshell 120 EC-C18 with longer column – still under 400 bar!
2.0 ml/min, 394 barmAU
20
40
60
80
100
min2 4 6 8 10 12
0
1. Hydroquinone2. Resourcinol3. Catechol4. Phenol5. 4-Nitrophenol6. p-cresol7. o-cresol8. 2-Nitrophenol9. 3,4 di methyl phenol10. 2,3 di methyl phenol11. 2,5 di methyl phenol12. 1-napthol
Conditions: Column: Poroshell 120 EC-C18, 4.6 x 100mm, 2.7umMobile Phase:Solvent A: Water with 0.1% Formic AcidSolvent B: AcetonitrileGradient::Time %B2.0 5%17 60%1200 SL controlled temperature at 25 °C2 uL flow cell
Page 19
Poroshell 120 150mm Columns – HPLC or UHPLC
2 mL/min
min0 2 4 6 8 10 12 14 16 18
mAU
0
20
40
60
80
100
120
min0 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. Hydroquinone2. Resourcinol3. Catechol4. Phenol5. 4-Nitrophenol6. p-cresol7. o-cresol8. 2-Nitrophenol9. 3,4 di methyl phenol10. 2,3 di methyl phenol11. 2,5 di methyl phenol12. 1-napthol
Gradient: 1mL/minTime%B6.0 5%51 60%Gradient: 2mL/minTime%B3.0 5%25.5 60%
Page 20
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Poroshell 120 Resists Plugging with 2 um Frit Challenging Samples - Plasma
Diflusinal in Plasma
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
340
360
380
400
1 501 1001 1501 2001 2501
Injections
Pre
ssur
e
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
Effi
cien
cy (N
)
End Press
Plates
Column: Poroshell 120 EC-C18, 3.0 x 50mm, 2.7um LC: Agilent 1200 RRLC (SL) Sample: Precipitated Plasma: 2 parts Plasma: 7 Parts 20/80 Water-MeCN w/0.1 % Formic Acid with 1 Part Diflusinal in 50/50 Water-MeCN 10 ug/ml (Final concentration Diflusinal 1 ug/ml) Shaken and allowed to settle 10 minutesNot Centrifuged/ Not FilteredInjection Volume: 1ul injections
Solvent A: Water w/0.1 % TFASolvent B: MeCN w/0.08 % TFAFlow Rate 1 ml/min 1 ul injectionTime % B0 200.5 900.6 901.1 202.5 20
Page 21
Superficially Porous Columns Can be Used to Meet USP Requirements
From USP list of LC column packings 4/2009
L
PACKING
BRAND NAME
MANUFACTURER
L1
Octadecyl silane chemically bonded to porous silica or ceramic micro-particles, 1.5 to 10 µm in diameter, or a monolithic rod
AminoQuant AQUASIL C18 Ascentis C18 Ascentis Express C18 ASI C18 ASI microC18
Agilent Technologies Thermo Scientific Supelco Supelco Analytical Sciences Inc. Analytical Sciences Inc.
L
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USP and FDA Method Adjustment CriteriaMethod Adjustment Criteria for Column Dimensions
Parameter Maximum Specifications Comments/Examples
Column Length ± 70%250mm 75mm150mm 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 VolumeReduce 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 SizeReduce 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 2009See Stimuli article in Pharmacopeial Forum 2009; 35(6)
Page 23
Comparison of 4.6 x 250 mm 5 um to Poroshell 120 EC-C18 4.6 x 100 mm, 2.7um
min5 10 15 20 25 30
mAU
0
20
40
60
80
100
9.7
12
11
.11
6
11
.59
6
12
.67
4
15
.24
8
16
.15
11
6.4
35
20
.68
7
23
.07
6
29
.29
0
min5 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
.55
8
5.4
50
5.9
20
7.0
37
� Faster analysis (shorter column higher flow rate)� More Sensitive (smaller particle sharpens peak)� Better Optimized (faster analysis allows better tweaking)� Both run on Agilent 1100 G1315B DAD (under 400 bar)� No change in sample prep required, 2 micron frit used on both columns
325 bar
110 bar
Sulfadiazine,Sulfathiazole SulfapyridineSulfamerazine,Sulfamethazine, Sulfamethazole, Sulfamethoxypyridazine,SulfachloropyridazineSulfamethoxazole, Sulfadimethoxine
Time %B0 833 3334 33Column: Eclipse Plus C184.6 x 250mm, 5umFlow Rate: 1 mL/min
Mobile Phase:A: 0.1% formic acid in WaterB: 0.1% formic acid in ACN
Time %B0 812 3313.2 33Column: 4.6 x 100mmPoroshell 120 EC-C18, 2.7umFlow Rate: 1 mL/min
Page 24
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Comparison of 4.6 x 250 mm 5 um to Poroshell 120 EC-C18 4.6 x 100 mm, 2.7um
min5 10 15 20 25 30
mAU
0
20
40
60
80
100
9.7
12
11
.11
6
11
.59
6
12
.67
4
15
.24
8
16
.15
11
6.4
35
20
.68
7
23
.07
6
29
.29
0
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
.55
8
5.4
50
5.9
20
7.0
37
325 bar
min
See conditions previous slide
Page 25
Chromatographic Optimization with Poroshell 120Same Process as on Other Columns
1. Adjust Injection volume proportional to column volume (5 ul to 2 ul).
2. Adjust Gradient Length proportional to column Length (100/250 x30 min) = 12 min
3. Optimize Flow Rate for Smaller Particles (adjust gradient time while keeping column volume constant) may improve resolution
4. Adjust gradient for selectivity differences
min0 2 4 6 8 10
mAU
0
20
40
60
80
100
3.7
06
4.3
71 4.5
12
4.8
77
5.9
29
6.3
75
6.4
72
8.0
38
8.9
82 1
1.5
32
min0 2 4 6 8 10
mAU
0
20
40
60
80
100
2.4
75
2.9
23 3.0
21
3.2
63
3.9
77
4.2
66
4.3
34
5.3
68
5.9
90 7
.70
5
min0 2 4 6 8 10
mAU
0
20
40
60
80
1.8
56
2.1
94 2.2
68
2.4
48
2.9
89
3.2
02
3.2
54
4.0
23
4.4
80 5
.77
6
min0 2 4 6 8 10
mAU
0
20
40
60
2.3
57
3.2
52
3.3
52
3.7
23
5.5
45
6.5
17
6.8
35
8.4
79
9.1
34
10
.51
6
min0 2 4 6 8 10
mAU
0
20
40
60
0.6
36
1.7
59
2.2
53
2.3
69
2.6
69
3.9
74 4.5
21
4.6
32
5.5
11
5.9
74
7.0
78
Adjust injection Volume and Gradient Length
Adjust Flow Rate Keeping Number of Column Volumes Constant (1 to 1.5)
Adjust Flow Rate Keeping Number of Column Volumes Constant (1.5 to 2)
#1, 2
#3
#3
#4
#4 Adjust Gradient for Selectivity Differences
Page 26
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Chromatographic Optimization with Poroshell 120Same Process as on Other Columns
4. Optimizing the Separation by Adjusting the Gradient
min0 2 4 6 8 10
min0 2 4 6 8
Adjust Gradient for Selectivity Differences
Page 27
How is a Poroshell 120 Particle Made?
Step 1 – Make the solid core
> Cores are a uniform size with a smooth surface for a tight overall particle size distribution
Step 2 – Apply the porous shell
> This is done in a single step – no multilayering
> A coacervation technique is used – and represents a unique process and significant innovation by Agilent for more consiste nt chromatography
Step 3 – React the bonded phase
> Two different C18 bonded phases are available – EC-C18 – endcapped C18 and SB-C18 – non-endcapped C18
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Step 1 – Make the Solid Core
Cores are a uniform 1.7um size with a smooth surface
RSD of seven batches of cores = 0.04%
This leads to a tight final particle size distribution
Tighter than with totally porous particles
SEM of Cores
Page 29
Comparison of Particle Size Distributions Between Totally Porous and Poroshell 120 Particles
Page 30
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Comparison of Particle Size DistributionsPoroshell 120 is 25% Narrower
Poroshell 120* 1.8 µm 3.5 µm 5.0 µm10% 2.50 µm 1.67 µm 3.07 µm 4.59 µm90% 2.91 µm 2.45 µm 4.44 µm 6.21 µm
90%/10% ratio 1.16 1.47 1.45 1.35
� The 1.8, 3.5 and 5um particles all have a normal particle size distribution (number below 1.5 for totally porous particles).� The Poroshell 120 particle size distribution is 20-25% narrower.� This narrower PSD improves column efficiency over a totally porous particle – i.e. impacts the “A” term in the Van Deemter equation.� This is one reason for the high efficiency of Poroshell 120
*Lot B10009
Page 31
Step 2 – Make the Porous Outer Layer
• The Agilent process for Poroshell 120 differs from other processes for superficially porous particles.
• A single step coacervation process is used, rather than a multilayering process.
• Coacervation is the process by which small sol particles come together to form the porous particle
• Like the finely controlled pore size/particle size process of ZORBAX
• 1 step reduces variability – more reproducible
Page 32
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Step 3 – Attach the Bonded Phase
2 C18 Bonded Phases are available
> Poroshell 120 EC-C18 – endcapped C18 for best peak shape
– Should be the first choice for most separations
– This includes peptide mapping with LC/MS compatible mobile phases
– Most similar to competitive superficially porous products (they are all endcapped)
> Poroshell 120 SB-C18
– Great alternate selectivity because non-endcapped
– Best performance at low pH, i.e. longest lifetime
Page 33
Specifications of Poroshell 120 Bonded Phase
•Poroshell 120 EC-C18
•Pore Size - 120Å
•Particle Size – 2.7um
•pH range – 2-9
•Endcapped
•Surface area – 130 m2/g
•Carbon load – 8%
•Surface coverage – 3.3 umol/m2
•Poroshell 120 SB-C18
•Pore Size - 120Å
•Particle Size – 2.7um
•pH range – 1-8
•Non-endcapped
•Surface area – 130 m2/g
•Carbon load – 7.5%
•Surface coverage – 2.1 umol/m2
Page 34
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Group/Presentation TitleAgilent Restricted
Poroshell 300 – Agilent’s First Poroshell Columns
High Resolution, Fast Separations of Polypeptides and Proteins
•Poroshell 300 columns are for the separation of large peptides and proteins
•Poroshell 300 particles have a solid silica core with a superficially porous surface with a 300Å pore size
•This results in more efficient mass transfer, sharper peaks and fast, efficient separations with large proteins and peptides
SOLID CORE
Porous Shell
5 um
Poroshell is more than Poroshell 120. It is choices for pharma and biopharma
Page 35
Group/Presentation TitleAgilent Restricted
High Flow Rates with 2.1 mm ID Poroshell for High Resolution and Fast Separations
12
34
5
67
8
0 0.5 1.0Time (min)
Columns: Poroshell 300SB-C182.1 x 75 mm, 5 um
Mobile Phase:A: 0.1% TFAB: 0.07% TFA in ACN
Gradient: 5 – 100% B in 1.0 min.Flow Rate: 3.0 mL/min.Temperature: 70°CPressure: 250 barDetection: UV 215 nm
Sample:1. Angiotensin II2. Neurotensin3. Rnase4. Insulin5. Lysozyme6. Myoglobin7.Carbonic Anhydrase8.Ovalbumin
Poroshell can provide high efficiency at higher flow rates for extremelyrapid separations of proteins and peptides. This is due to more rapid mass transfer of the superficially porous particle
Page 36
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Poroshell 120 vs. Poroshell 300
Use Poroshell 120 for peptide mapping
> Peptide digests of proteins or monoclonal antibodies
> Typical gradient separations work well
> Use flow rates of 0.5 mL/min or higher for fast separations on 2.1 mm ID columns
> Use Poroshell 120 EC-C18 for LC/MS applications with formic acid
Use Poroshell 300 for larger biomolecules
> Does the sample contain all polypeptides or larger
> Can be used with very large proteins
> Use at a high flow rate for optimum efficiency (i.e. 1.0mL/min on 2.1 mm ID columns)
Page 37
Agilent 1290 + 6530 Accurate Mass Q-TOF: Rapid Peptide Mapping of IgG
TIC
ECC for Matched Peptides
15 minute runPoroshell 120 2.1x 150 mm column
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20
Pressure Comparison on Superficially Porous and Sub 2-um
Page 39
0
100
200
300
400
500
600
700
0 2 4 6 8 10 12 14
Pre
ssur
e (b
ar)
u (mm/s)
Pressure
Eclipse Plus C18, 3.0 mm x 100 mm, 1.8 um (USUYB01453)
Poroshell 120 EC-C18, 3.0 mm x 100 mm, 2.7 um (USCFX01009)
Ascentis Express C18, 3.0 mm x 100 mm, 2.7 um (USKJ001754)
Phenomenex Kinetex C18, 4.6 mm x 100 mm, 2.6 um (501268-43)
Acetonitrile/Water (60/40)
Phenmomex Kinetex has a higher pressure because of a smaller particle size.
Comparison of Peak Shape on Superficially Porous Columns
min0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
mAU
0
20
40
60
80
KinetexAscentis Express
Poroshell 120
Poroshell 120 Tf = 1.48Ascentis Express Tf = 2.68Kinetex Tf = 4.78
Columns: 4.6 x 50mm, Mobile Phase: 20 mM 40% Na2HPO4, pH 7.00 60% Acetonitrile Flow Rate: 1.5 mL/minSample: 2 uL injection of 250 ug/mL amitriptyline, 50 ug/mL uracil in H2O/CH3CN (9:1) Temp: 24°C Detector 254nm, 2-uL flow cell
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21
min1 2 3 4 5
mAU
0
100
200
300
400
5000.
234
0.29
8
0.41
2
0.54
4
0.72
40.
803
1.01
3
1.49
2
2.27
3
3.55
1
min1 2 3 4 5
mAU
0
100
200
300
400
500
0.21
50.
269
0.37
4
0.49
9
0.67
00.
740
0.95
1
1.42
5
2.21
2
3.51
7
Poroshell 120 Maintains More Efficiency at Higher Flow Rates
Page 41
Flow Rate: 1.665 mL/min
Acetonitrile/Water (60:40)Injection Volume: 4.00 µLTCC: 26 oCDAD: Sig=254,4nm Ref=360,100nm
Sample: RRLC Checkout Sample (PN 5188-6529) spiked w/ 50 µL 2 mg/mL Thiourea in water/acetonitrile (65:35)
← N = 19929Press = 485 bar
← N = 16551Press = 480 bar
Poroshell 120 EC-C18, 3.0 x 100 mm, 2.7 um
Ascentis Express C18, 3.0 x 100 mm, 2.7 um
Peak Capacity of Poroshell 120 is slightly lower than RRHT but at a lower pressure
Using Agilent 1200SL w/80 Hz Detection
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
0 0.5 1 1.5 2 2.5 3 3.5 4
Pea
k C
apac
ity
Flow Rate (ml/min) scaled gradient
Average Peak Capacity
Poroshell 120 EC C18
Competitor C18 2.5 um
Eclipse Plus C18 5 um
Eclipse Plus 3.5 um
Eclipse Plus C18 1.8 um
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22
Peak Capacity (Pc) of RRHT is Higher than Superficially Porous Columns
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
0 0.5 1 1.5 2 2.5 3 3.5 4
Pea
k C
apac
ity
Flow Rate (ml/min) Scaled gradients
Average Peak Capacity
Poroshell 120 EC C18
Ascentis Express C18
Kinetex C18
Eclipse Plus C18 1.8 um RRHT
Page 43
Peak Capacity Comparison on Poroshell 120 and Competitive Products
1. Hydroquinone2. Resourcinol3. Catechol4. Phenol5. 4-Nitrophenol6. p-cresol7. o-cresol8. 2-Nitrophenol9. 3,4 di methyl phenol10. 2,3 di methyl phenol11. 2,5 di methyl phenol12. 1-napthol
All compounds prepared at 1 mg/ml (prepared at 0.1 mg/ml) in water.Compounds were combined in equalvolumes to yield approximately 0.1 mg/ml 10ul injection volume , 4.6 x 50 mmA: 0.1 % Formic Acid in WaterB: Acetonitrile
270 nm, no reference
Poroshell 120 EC C182 ml/min 220 BarPc Ave=54
Ascentis Express C182 ml/min 210 BarPc Ave=55
Eclipse Plus C18 RRHT2 ml/min 364 BarPc Ave=61
Kinetex C182 ml/min 232 BarPc Ave=50 2 ml/min
Time % B1 1 52 8.5 603 10 604 11 5
min0 1 2 3 4 5 6
mAU
0
10
20
30
40
50
60
0.35
6
0.70
7
1.34
9
1.81
5
3.04
4 4.17
1
4.41
0
4.54
1
4.86
3
5.28
6
5.51
95.
605
6.14
4
min0 1 2 3 4 5 6
mAU
0
10
20
30
40
50
60
70
0.30
4
0.56
1
1.07
8
1.48
0
2.73
6
3.86
3
4.12
4
4.25
5
4.61
9
4.99
9
5.23
15.
316
5.85
6
min0 1 2 3 4 5 6
mAU
0
10
20
30
40
50
60
0.34
8
0.76
4
1.53
1
2.04
2
3.30
9
4.38
8
4.62
6
4.78
1
5.17
7
5.47
9
5.72
75.
809
6.31
7
min0 1 2 3 4 5 6
mAU
0
10
20
30
40
50
60
70
0.33
2
0.63
2
1.09
7
1.37
9
2.58
4
3.86
2
4.00
7
4.12
3 4.46
0
4.90
8
5.12
05.
204
5.81
9
Columns: 4.6 x 50mm Flow Rate: 2 mL/min
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23
Which Agilent Product Do You Recommend? Sub 2um or Poroshell 120For an HPLC recommend Poroshell 120
> Unless the chromatography requires scale-up in particle sizes
For a UHPLC both choices are possible
> Consider the goals of the customer and applications available
> Sub 2um will be an excellent choice for the highest pressure UHPLCs
Page 45
Agilent Has Three Types of Columns for UHPLC
There are 3 column types for UHPLC – choice depends on your requirements
RRHT columns – 1.8um columns for fast LC up to 600 bar > Designed for compatibility with the Agilent RRLC
> Compatible with some UHPLC’s with pressure limits up to 800 bar
RRHD columns – 1.8um columns for up to 1200 bar> Designed for compatibility with the new Agilent 1290 Infinity LC
> Useable up to 1200 bar
> Fully UHPLC compatible
Poroshell 120 columns> 600 bar pressure limit for use with HPLC/UHPLC
> UHPLC type performance but excellent fit for 400 ba r LC’s
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Agilent UHPLC Column Selection Guidelines
Fast Analysis
High Rs (N)
400 bar LC “Fitness”
1000+ bar UHPLC
“Fitness”
Scalability of Particle1.8, 3.5um
etc.
ID’s 4.6, 3.0 & 2.1 mm
Dirty Samples?
Poroshell 120, 600
bar � � � � X only 2.7um� 2um �frit
RRHT, 600 bar � � �<50mm � � � �RRHD,
1200 bar � � X � � X (3.0 & 2.1)
�Page 47
Group/Presentation TitleAgilent Restricted
Month ##, 200X
Sub 2um vs. Poroshell 12017 Amino Acid Analysis on 1290 Infinity
min0 2 4 6 8 10 12
mAU
0
50
min0 2 4 6 8 10 12
mAU
0
50
min0 2 4 6 8 10 12
mAU
0
50
Poroshell 120 EC-C183.0 x 100mm, 2.7um
Pmax=331 barF=0.86 mL/min
RRHT Eclipse Plus C183.0 x 100mm, 1.8um
Pmax=474 barF=0.86 mL/min
RRHD Eclipse Plus C183.0 x 100mm, 1.8um
Pmax=528 barF=0.86 mL/min
Rs6,5=1.98
Rs6,5=2.28
Rs6,5=2.90
Rs14,13=1.81
Rs14,13=2.34
Rs14,13=1.81
W½ 9 =0.043 min.
W½ 9 =0.036 min.
W½ 9 =0.045 min.
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Environmental Phenols
min0 2 4 6 8 10
mU
0
20
40
60
80
min0 2 4 6 8 10
mAU
0
20
40
60
80
)
Eclipse Plus C18RRHT 483 Bar
Poroshell 120 EC C18 332 Bar
A: Water 0.1 % Formic AcidB: Acetonitrile 0.1 % formic Acid 10µl injGradient 2 ml/minInitial 8 % B10 min 30% B275 nm 2mm flow cellAgilent 1200 SL 40 CNo pulse damperNo mixer 3 ul heater4.6 x 100 mm 2.7 or 1.8µm
1. Hydroquinone2. Resorcinol3. Catechol4. 4-Nitrophenol5. p-cresol6. o-cresol7. 2-Nitrophenol8. 2,3 Dimethyl phenol9. 2,5 Dimethyl phenol10. 1 Naphthol
12
4 5
67,8
910
11
12
12
3
12
34 5
67,8
910
11
Theory of Poroshell – Why Choose This Approach to Fast LC?What do we normally do?
What are our options?
Poroshell impact on large molecules
Poroshell application to small molecules
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Van Deemter – The Theory Behind
Theo
retica
l Plat
e heig
ht H
linear flow u
Multipath Term
Resulting Van-Deemter curve
Resistance to Mass Transfer
H = A + B/u + C x ularge particle small particle
The smaller the theoretical plate height H, the better the Resolution!
u opt
H minLongitudinal Diffusion
C term – resistance to mass transfer is improved by •Reducing particle size – this is the current standard approach •Improving diffusion
Page 51
What Parts of the Knox/VanDeemter Equation Are Affected by the Column ParticleA term – eddy diffusion and flow distribution
> Yes, this is impacted by the column particle
> Column particle size and column packing quality impact this
> A tight particle size distribution improves the A term
B term – longitudinal diffusion > Yes, this is impacted by column particle
> This is impacted by column pore size
C term – mass transfer component> Yes, this is impacted by the column particle and its properties
> Mass transfer is improved by using smaller particles with shorter diffusion paths
> This is improved with a superficially porous particle
> This allows high efficiency at high flow rates
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What makes Poroshell 120 different from other SPP (Superficial Porous Particle) columns?• The coacervation process used to make the
porous outer layer in just one step, rather than a multilayering process, reduces variability –more reproducibility
• The narrowest PSD (particle size distribution) wiht a 90%/10% ratio of 1.16, provide the best efficiency.
• Best peak simetry in the market. TF closest to 1, better than the competition
May 3, 2010Confidentiality Label
53
SummaryPoroshell 120 is a new LC column that complements the productivity solutions from Agilent
> It is a New Approach to High Resolution and High Speed LC, which can be used in any HPLC or UHPLC
> High Efficiency Separations can be achieved on Poroshell 120 with Low Pressure .
> Can be used with dirty samples, without seing a high increase in pressure
> Can be used on an USP method without compromising the Method Adjustment Criteria
> The coacervation process used to make the porous outer layer and the narrowest PSD, gives to Poroshell 120 the best reproducibility, the better peak shape, the highest efficiency, the lower pressure and the highest peak capacity among all the superficially porous columns in the market
Page 54