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Rapid Resolution -Practical Considerations -Practical Considerations -
Series 6
LC Columns and Consumables
Ed KimApplication EngineerMay 6, 2009
What are the Advantages of Sub 2-Micron Columns?Columns?1. Faster analyses – up to 10 – 20X faster
Small particles generate equivalent efficiency in a shorter column p g q ylengthThis allows equivalent resolution in less time – or faster analyses
2. Higher resolution analyses in equivalent or less time2. Higher resolution analyses in equivalent or less time
Improved resolution can be critical to some analysesLonger columns with small particles can provide extremely high resolution or peak capacityresolution or peak capacitySmaller particles generate more efficiency than larger particles in the same length column
3 Hi h iti it3. Higher sensitivity
4. Lower costs – time, solvent etc.
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What Are the “Limitations” of Sub 2-micron Columns? or
“Are They Easy Enough to Use?”Are They Easy Enough to Use?
1. Backpressure and particle size distribution – is there a balance?
2. Are there enough choices to do the separation you want?
3. What extra preparation/care do you need to take to use the columns f ll ?successfully?
4. Are there sample limitations for these columns?
5 Can you use the same range of mobile phases and conditions5. Can you use the same range of mobile phases and conditions (temperature etc.) with sub 2-micron columns?
6. What LC instrumentation is required to achieve maximum benefit from sub 2-micron columns?
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Advantages – Faster Analyses
What makes the analysis faster?• Theory – van Deemter curves
Are faster analyses easily achieved?• Theory/Practice
What does it take to go 5X, 10X, or 20X faster?
Are there practical examples to go with this?Are there practical examples to go with this?
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van Deemter – The Theory BehindFor small molecules, smallerFor small molecules, smaller particles result in faster diffusionResult: faster chromatography
ate
heig
ht
l ti l ll ti l
The smaller the theoretical plate height H, the better the Resolution!
oret
ical
Pla
Resulting Van-Deemter curve
H = A + B/u + C x ularge particle small particle
Theo
H
curve
Resistance to Mass Transfer
H min
Multipath Term
Longitudinal Diffusion
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linear flow uu opt
van Deemter Curves1.8um Rapid Resolution HT Columns1.8um Rapid Resolution HT Columns
0.0045
1 8 RRHT
Higher Speed – Higher Resolution – Higher Sensitivity
0 0030
0.0035
0.0040 Columns: ZORBAX Eclipse XDB-C18Dimensions: 1.8 μm, 4.6 x 50mmEluent: 85:15 ACN:WaterFlow Rates: 0.05 – 5.0 mL/min
1.8um RRHT columns provide flow rate independent efficiency gain!
0.0020
0.0025
0.0030
(cm
/pla
te)
Temp: 20°CSample: 1.0μL Octanophenone in Eluent
5.0 μm
efficiency gain!
Therefore use faster flow rates for faster analyses!
0.0005
0.0010
0.0015
HET
P
3.5 μm
1 8 m
analyses!
-0.0005
0.0000
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
1.8 μm 260,740 N/m @ 2mL/min 5.0 mL/min
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Interstitial Linear Velocity (ue- cm/sec)
All scaling calculations to transfer methods to RRHT, 1.8um particles are done using the Agilent MethodTranslator
For free –P/N 5989-5130EN
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Contributed Example Shows Minimum 4X Faster Method
Compounds A-I monitored4X shorter, with no loss in RsRs of all minor compounds
…maintained or improved
35 min.
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9 min
10X Shorter Analysis Time with a 4.6 x 50mm RRHT Column – Same Method, Shorter ColumnRRHT Column Same Method, Shorter Column
RRHT, SB-C184.6 x 50mm
15 min 1.5 min
QA/QC Analyses are sped up 10XNo change in resolution – system suitability analysis the sameSensitivity improvedSame mobile phase, other conditions used
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USP Methods with Rapid Resolution HT Columns –10X Faster with Shorter Columns10X Faster with Shorter Columns
2
USP Analysis of GuaifenesinSample: 1. Guaifenesin – 0.04 μg/μL, 2. Benzoic Acid – 0.10 μg/μL
USP L1 Column: ZORBAX Eclipse XDB-C184.6x250mm, 5μm
Eluent: 40% Methanol:60% Water:1.5% Glacial Acetic AcidFlow: 1.0 mL/min, Injection: 8μL, Temp: 25 °C
1 Peak Tr N Rs1 6.63 12,737 02 11.19 18,552 15.8
min0 2 4 6 8 10 12
12 Rapid Resolution HT Column (L1)
ZORBAX Eclipse XDB-C18, 4.6x50mm, 1.8μmEluent: 40% Methanol:60% Water:1 5% Glacial
Peak TR N Rs 1 1.40 11,421 0
Key Parameters-Changed Column Length
min0 2 31
Eluent: 40% Methanol:60% Water:1.5% Glacial Acetic AcidFlow: 1.0 mL/min, Injection: 2μL, Temp: 25 °C
,2 2.33 12,909 12.3
gParticle Size
Key Parameters-SameFlow RateEluent
12
Rapid Resolution HT Column (L1)ZORBAX Eclipse XDB-C18, 4.6x30mm, 1.8μmEluent: 40% Methanol:60% Water:1.5% Glacial Acetic AcidFlow: 1.0 mL/min, Injection: 2μL, Temp: 25 °C
Peak TR N Rs 1 0.85 5,855 02 1.43 7,300 8.6
Injection SizeTemperature
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min0 21
Flow: 1.0 mL/min, Injection: 2μL, Temp: 25 C
Rapid Resolution HT Columns – Up to 20X Faster at High Temperaturesat High Temperatures
Up to 20x faster than conventional HPLC • With same or better performance (resolution, precision, sensitivity, carry over)
HPLC, 40°CSB-C184.6 x 150mm, 5µm1 20 l/ i 40°C
• Compliant with strictest regulatory performance requirements
RRHT SB-C182 1 50 1 8 RRLC 40°C
1.20ml/min, 40°CAnalysis Time = 11min
min0 2 4 6 8 10
2.1mm x 50mm 1.8µm1.00ml/min, 40°CAnalysis Time= 1.1min
RRLC, 40 C10x faster
min0.2 0.4 0.6 0.8 10
RRHT SB-C182.1mm x 50mm 1.8µm2.40ml/min, 95°CAnalysis Time: 0.4min
RRLC, 95°C27x fasterPW = 197msec
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0.2 0.60 0.4 min
What are the Advantages and Limitations of Higher Resolution Analyses?Higher Resolution Analyses?Advantages• More certainty that everything is resolved.• High peak capacity for analyzing complex samples.
LimitationLimitation• Longer RRHT columns will generate more back pressure and require
the RRLC
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Longer Eclipse Plus RRHT Columns Improve Efficiency Achieve Baseline Resolution from 0Efficiency Achieve Baseline Resolution from 0
Rs5,4 = 0 4.6 x 50 mm 1.8um Mobile phase: (69:31) ACN: waterFlow 1.5 mL (/min.Temp: 30 C
S Q S
1200000 1 25
186
Rs 1 384 6 x 100 mm 1 8um
Detector: Single Quad ESI positive mode scan
min2 4400000
3186bar
Rs5,4= 1.38
Rs = 1 61
4.6 x 100 mm 1.8um1600000
326bar
Rs5,4= 1.61
4.6 x 150 mm 1.8um1800000
2 4 6 8200000
12
5
413
Sample: 1. Anandamide (AEA) 2. Palmitoylethanolamide (PEA) 3. 2-arachinoylglycerol (2-AG) Rs5,4 = Baseline
min2 4 6 8 10
400000
34
413bar
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4. 1(3)-arachidonylglycerol5. Oleoylethanolamide (OEA)Baseline
Greater Peak Capacity Means More Efficiency and More Certain SeparationsMore Certain Separations
7 ImpuritiesAll 7 Baseline S t d!
4 Impurities2 Not Baseline Separated!
Customer Example Isocratic Impurity Method
Separated!Zoom of critical time range @ 7min
4.6 x 150, 1.8um4.6 x 150, 5um93 bar
490 barN = 28669
93 barN = 7259
More Peak Capacity Means Greater Certainty Everything is Resolved!!
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More Peak Capacity Means Greater Certainty Everything is Resolved!!
Standards - Improve Resolution with the Same Column Selectivity - Smaller Particle Size (1.8um)Column Selectivity Smaller Particle Size (1.8um)
Column: SB-C18, 4.6 x 50 mm Mobile Phase A= 0.1% formic, B=0.1% formic in MeCN (92:8) Flow Rate =1.5mL/min Inj. Vol: 2 ul Detection: UV 254 nm Flowcell: 2uL, 3 mm flow path
1. 1-methylxanthine2. 1,3-dimethyluric acid3. 3,7-dimethylxanthine
(theobromine)4. 1,7-dimethylxanthine
mAU
30405060 5 µm α2,1= 1.36
α3,2= 1.27
min0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.80
1020 α4,3= 1.79
mAU
20
30405060 1.8 µm
α2,1= 1.36
α3,2= 1.27
α = 1 79
min0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.80
1020 α4,3= 1.79
The selectivity is the same but resolution of standards is improved.
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Will actual samples containing theobromine look good?
RRHT Columns are for Real Samples! Samples Prepared and Filtered onlyPrepared and Filtered only
theobromine Hot Cocoa MixTheobromine is dominant in chocolate and not in tea (as expected).
caffeine
min0.5 1 1.5 2 2.5 3
Chocolate syrup
Tea bagtheobromine
min0.5 1 1.5 2 2.5 3
min0.5 1 1.5 2 2.5 3
Column: SB-C18, 4.6 x 50 mm Mobile Phase A= 0.1% formic, B=0.1% formic in MeCN (92:8) Flow Rate =1.5mL/min
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, , ( )Inj. Vol: 2 ul Detection: UV 254 nm Flowcell: 2uL, 3 mm flow path
Comparison of Peak Capacity with Change in Particle SizeParticle Size
mAU
100
120RRHT Eclipse XDB-C18,
2.1 x 100mm, 1.8µ, 600 Bar
Uracil35% increase in peak capacity!!
20
40
60
802.1 x 100mm, 1.8µ, 600 Bar
C-14 AlkylphenonePeak Capacity = 461 (n=10)
min0 2 4 6 8 10 12 14 16 180
20
mAU100
Rapid Resolution Eclipse XDB-C18,
40
60
80 2.1 x 100mm, 3.5µ, 400 Bar
Peak Capacity = 343 (n=10)
min0 2 4 6 8 10 12 14 16 180
20
Conditions: Column: as shown Mobile Phase: A:water B: ACN Gradient: 50 – 100% B in 20 minutes Flow Rate: 0.5 mL/min
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Injection Volume: 2 uL Sample: alkylphenones C3-C10,12,14 Temperature: 40°C Detection: 254 nm (80 Hz)
Particle Size - More Peak Capacity with 1.8um RRHT Columns - Peptide Map of BSARRHT Columns Peptide Map of BSA
673Peak CapacityColumn used
SB C18 2 1 150 1 8
mAU
40
50
Conditions: Columns: as listed, Mobile Phase: A:0.1% TFA in Water B:0.08% TFA in ACN Gradient: 5% B to 60%B in 25 min. Temperature: 80°C Sample: BSA tryptic digest
StartingPressure380 bar673SB-C18, 2.1x150mm, 1.8µ
10
20
30
35% More peak capacity, more resolution
380 bar
min14.5 15 15.5 16 16.5 17 17.5 18 18.5 19
-10
0
mAU
502SB-C18, 2.1x150mm, 3.5µ40
60
Less peak capacity less resolution
StartingPressure105 barPeak Capacity
0
20
Less peak capacity, less resolution
S ll ti l i h k t k it
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min15 16 17 18 19
-20 Smaller particle size = sharper peaks = greater peak capacity
Time Savings, Scalability and Improvement in Sensitivity with RRHT Eclipse Plus – 4.6 mm IDSensitivity with RRHT Eclipse Plus 4.6 mm IDColumns: Eclipse Plus C18, as described below. Mobile Phase: A: water, B: MeOH, (15:85)Temperature: 25°C Flow: 1 mL/min. Detection: 310, 4 nm, 0.5 s response time, semi-micro flow cell, Sample: Sunscreens
1.8 um Rs3,2= 6.51 Rs same,
4.6 x 50 mm, 1.8 µm
4.6 x 150 mm, 5 µmP=82 bar 5um Rs3,2= 6.41mAU
150
scalability demonstrated
P=208 bar
23100
Time savings, improved productivity with
Pressure below 400 bar, try before buy
1. 2-hydroxy-4-methoxybenzophenone
2. Padimate-O3 2 ethylhexyl trans 4
0
50
productivity with RRHT
3. 2-ethylhexyl trans-4-methoxycinnamate
4. 2-ethylhexyl salicylate8 10 12 min2 4 6
This blow-up of a chromatogram shows the added sensitivity achieved with a 1.8um column.
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p g yThe chromatograms are on the same scale and we are looking at the same 2 peaks in each.
Questions on Uses and Limits of Sub 2-micron columns?columns?1. Particle size distribution and backpressure – can you lower
the backpressure and not compromise results?
2. Are there enough choices to do the separation you want to do?
3. What extra preparation/care do you need to take to use the columns successfully?
4 Are there sample limitations for these columns?4. Are there sample limitations for these columns?
5. Can you use the same range of mobile phases and conditions (temperature etc.) with sub 2-micron columns( p )
6. What LC instrumentation is required to achieve maximum benefit from sub 2-micron columns?
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Particle Size Distribution for LC Columns
Particle Size Distribution should have a ratio of smallest to largest of 1:2, 1:1.5 would be ideal based on 90:10, 10:90 ratios
This is because:• Smallest particles, or fines, produce a greater flow resistance – or higher
b kback pressure• Therefore reducing fines is critical to lower back pressure on any
particle size distribution • Largest particles determine maximum efficiency and increase band
broadening A particle size distribution occurs in the manufacturing process• Number average particle size • Volume average particle size – always larger than the number-average
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Actual Example of Particle Size Distribution on ZORBAX 1.8um ParticlesZORBAX 1.8um Particles
Number Average Plot of ZORBAX 1.8 um materialDifferential Number
2500
3000
3500 Number StatisticsMean: 1.740 umMedian: 1.727 umMode: 1.732 um
1500
2000
2500
Num
ber Mode: 1.732 um95% Conf. Limits: 1.739 – 1.742 umSD.: 0.271 um
%> 10 90 Ratio
500
1000um 2.04 1.47 1.39
Small number of “off median” particles visible in number average plot
6543210
Particle Diameter (μm)
g p
PSD is “ideal” in terms for largest:smallest particles for high efficiency
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PSD is ideal in terms for largest:smallest particles for high efficiencyLow fines means lowest back pressure.
Actual Example of Particle Size Distribution on ZORBAX 1.8um ParticlesZORBAX 1.8um Particles
Volume Average Plot of ZORBAX 1.8 um materialDifferential Volume
8000
10000
3 )
Volume StatisticsMean: 1.889 umMedian: 1.805 umM d 1 761
4000
6000
lum
e (u
m3 Mode: 1.761 um
95% Conf. Limits: 1.887 – 1.892 umSD.: 0.401 um
%> 10 90 Ratio
2000
4000
Vo
%> 10 90 Ratioum 2.23 1.57 1.42
Volume Average Plot shows “off median” particles in distribution due to particle
Particle Diameter (μm)1 2 3 4 5 6
0
particles in distribution, due to particle engineering
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(μ )
High Performance - Van Deemter Curves for Narrow vs. Production Particle Size Distribution 1.8μm SB-C18μ
9
10Columns: ZORBAX SB-C18Dimensions: 4 6 x 30mm 1 8 μm
h vs. υ Fit CoefficientsProduction PSD Narrow
7
8
ight
(h)
Dimensions: 4.6 x 30mm, 1.8 μmEluent: 85:15 ACN:WaterFlow Rates: 0.05 – 5.0 mL/minTemp: 20°CSample: 1.0μL Octanophenone in EluentD t ti UV 245
Production PSD Narrow PSDVD A: 0.795 0.760
B: 3.97 4.26C: 0 085 0 077
4
5
6
d Pl
ate
Hei
7793 Plates @1.6 mL/min
8065 Plates @
Detection: UV 245nmC: 0.085 0.077r2: 0.9994 0.9998
1
2
3
Red
uced Production 1.8μm – Optimum PSD
Narrow 1.8μm – Narrow PSD
(-3.4%)@
2.0 mL/min
R ld f 2 0 t 1 96!
0
1
0 2 4 6 8 10 12 14 16 18 20
Narrow 1.8μm Narrow PSDRs would go from 2.0 to 1.96!This is not noticeable in practice!
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Reduced Linear Velocity (ν)
Lower Back Pressure for Production vs. Narrow Particle Size Distribution 1.8μm SB-C18Particle Size Distribution 1.8μm SB C18
250
300
Narrow 1.8μm – Narrow PSDColumns: ZORBAX SB-C18Dimensions: 4.6 x 30mm, 1.8 μmEluent: 85:15 ACN:Water
200
250
ar)
Eluent: 85:15 ACN:WaterFlow Rates: 0.05 – 5.0 mL/minTemp: 20°CSample: 1.0μL Octanophenone in EluentDetection: UV 245nm
100
150
ress
ure
(Ba
25% HIGHER Pressure at Optimum Flow (2.0 mL/min)
Production 1.8μm – Optimum PSD
Lower Pressure
50
100Pr
System Pressure (No Column)
00.0 1.0 2.0 3.0 4.0 5.0
Flow Rate (mL/min)
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Flow Rate (mL/min)
1. The Complete List of RRHT 600 bar ColumnsEclipse
Dimensions Eclipse Plus C18 Eclipse Plus C8 XDB-C18 Eclipse XDB-C8 Extend-C184.6 x 150 959994-9024.6 x 100 959964-902 959964-906 928975-902 928975-906 728975-9024.6 x 50 959941-902 959941-906 927975-902 927975-906 727975-9024.6 x 30 959931-902 959931-906 924975-902 924975-906 724975-9024.6 x 20 926975-902 926975-906 726975-9023 0 x 150 959994 302
2 Recommended Starting Choices:
3.0 x 150 959994-3023.0 x 100 959964-302 959964-306 928975-302 928975-306 728975-3023.0 x 50 959941-302 959941-306 927975-302 927975-306 727975-3023.0 x 30 924975-302 924975-306 724975-3023.0 x 20 926975-302 926975-306 726975-3022.1 x 150 959794-9022.1 x 100 959764-902 959764-906 928700-902 928700-906 728700-902
• 959941-902• 959741-902They are Eclipse Plus C18 Columns.2.1 x 50 959741-902 959741-906 927700-902 927700-906 727700-902
2.1 x 30 959731-902 959731-906 924700-902 924700-906 724700-9022.1 x 20 926700-902 926700-906 726700-902Dimensions SB-C18 SB-C8 SB-Phenyl SB-CN SB-AQ Rx-Sil4.6 x 150 829975-902 829975-906 829975-912 829975-905 829975-9144.6 x 100 828975-902 828975-906 828975-912 828975-905 828975-914 828975-9014 6 x 50 827975 902 827975 906 827975 912 827975 905 827975 914 827975 901
C18 Columns.
4.6 x 50 827975-902 827975-906 827975-912 827975-905 827975-914 827975-9014.6 x 30 824975-902 824975-906 824975-912 824975-905 824975-9144.6 x 20 826975-902 826975-9063.0 x 150 829975-302 829975-306 829975-312 829975-3053.0 x 100 828975-302 828975-306 828975-312 828975-305 828975-314 828975-3013.0 x 50 827975-302 827975-306 827975-312 827975-305 827975-314 827975-3013.0 x 30 824975-302 824975-306 824975-3053.0 x 20 826975-302 826975-3062.1 x 150 820700-902 820700-906 820700-912 820700-9052.1 x 100 828700-902 828700-906 828700-912 828700-905 828700-914 828700-9012.1 x 50 827700-902 827700-906 827700-912 827700-905 827700-914 827700-9012.1 x 30 824700-902 824700-906 824700-912 824700-905 824700-9142.1 x 20 826700-902 826700-906
http://www chem agilent com/Scripts/PDS asp?lPage=9818 (Main product page)
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http://www.chem.agilent.com/Scripts/PDS.asp?lPage=9818 (Main product page)http://www.chem.agilent.com/Scripts/Generic.ASP?lPage=11424&indcol=N&prodcol=N (P/N List)
Are all the C18’s Different Selectivity Choices?
Eclipse Plus C18
M bil h (69 31) ACN t
1 23
41st choiceBest Resolution& Peak Shape
StableBondSB C18
Mobile phase: (69:31) ACN: waterFlow 1.5 mL/min.Temp: 30 °CDetector: Single Quad ESI positive mode scan Columns: RRHT
1 2 3 4 5
1 234
p
2nd choiceGood alternate selectivity due t d d
Eclipse
SB-C18 Columns: RRHT 4.6 x 50 mm 1.8 um
Sample:1. anandamide (AEA)2 P l it l th l id (PEA)1 4
min1 2 3 4 5
to non-endcapped
3rd choice EclipseXDB-C18
2. Palmitoylethanolamide (PEA)3. 2-arachinoylglycerol (2-AG)4. Oleoylethanolamide (OEA)
1 23
4
1 2 3 4 5
3 choiceGood efficiency & peak shapeResolution could be achieved
4th choice Each C18 offers something
Extend-C181 2,3 4
4th choiceResolution not likely,Other choices better, for this separation.
unique.While Eclipse Plus C18 might be the best general choice, other separations may be
better with others
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1 2 3 4 5
better with others.
More RRHT Bonded Phase Choices Allow for Optimized Method Development
mAU
400
450
5 Choices in SB family can be used for method optimizationRRHT SB-Phenyl best choice - more resolution in half the time!
2X the needed timeSB-C18 1.8um
300
350
Not the best choice!
Change in Elution Order Peaks 3,4 & 5
S lSB-Phenyl 1.8um
SB-C8 1.8um
200
250
Best Choice!Sample:1. Tolmetin2. Naproxen3. Diflunisal4. Ibuprofen5 Diclofenac
SB Phenyl 1.8um
50
100
150
Conditions: Columns: RRHT 4.6 x 50mm, 1.8um Mobile Phase: Acetonitrile (40%):Buffer 60% pH=2.4 25mM NaH2PO4
5. DiclofenacFor SB-PhenylSB-CN 1.8um
min0 1 2 3 4 5
0
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Flow Rate: 1.85 mL/min Detection: DAD, 254 nm LC: Agilent 1200 RR LC Sample: As listed, 1 uL injection
How To “Match” a Column to a ZORBAX RRHT Column
General Phase Type Starting ZORBAX Choice
Typical “endcapped” C18 or C8 bonded phases, newer columns Eclipse Plus C18 or C8
Endcapped C18 or C8 columns olderEndcapped C18 or C8 columns, older generation Eclipse XDB-C18 or C8
Non-endcapped columns StableBond C18
Older types of columns StableBond C18, C8 etc.
Aqueous “type” columns SB-AQ
CN or Phenyl SB-CN, SB-Phenyl
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2. What Extra Precautions Do You Need to Use RRHT Columns Successfully?RRHT Columns Successfully? 1. At column installation
Solvent changesgLC System cleanlinessColumn Equilibration
2 Column use2. Column useAre you operating at the pressure you anticipate?Check your gradient
3. Mobile phaseAre there limitations?
4 S l id ti4. Sample considerationsParticulate free samplesInjection solvent
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j
RRHT Column Installation Recommendations1 Purge the pumps (connections up to the column) of any buffered mobile phases1. Purge the pumps (connections up to the column) of any buffered mobile phases.
Flush at least 5 mL of solvent before attaching the column to instrument. Goal: Eliminate any dried out or precipitated buffer from the system so it doesn’t wash onto the column and plug the frit.
2. Flush the column with your mobile phase (compatible with the solvents the column was shipped in) starting slowly at 0.1 mL/min for a 2.1 mm ID column, 0.2 mL/min for a 3.0 mm ID column, and 0.4 mL/min for 4.6 mm ID. Goal: Avoid a pressure spike when the new mobile phase reaches the column. This p p poccurs when the different solvents mix. The low flow rate allows this to happen without causing an unanticipated pressure change.
3. Increase the flow rate to the desired flow over a couple of minutes. Goal: anticipate the final operating pressureanticipate the final operating pressure
4. Once the pressure has stabilized, attach the column to the detector.
5. Equilibrate the column and detector with 10 column volumes of the mobile phase i t G l d ibl h t h f th 1stprior to use. Goal: reproducible chromatography from the 1st run
6. If you are running a gradient, check that the pressure range of the gradient –which may be 100 – 130 bar or more, will not cause the system to overpressure, before starting any sequence Goal: no surprises good unattended operation
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before starting any sequence. Goal: no surprises, good unattended operation over 100’s or 1000’s of injections.
RRHT Column Installation Recommendations (cont.)(cont.)4. Once the pressure has stabilized, attach the column to the detector.
5. Equilibrate the column and detector with 10 column volumes of the5. Equilibrate the column and detector with 10 column volumes of the mobile phase prior to use. Goal: reproducible chromatography from the 1st run
6 If you are running a gradient check that the pressure range of the6. If you are running a gradient, check that the pressure range of the gradient – which may be 100 – 130 bar or more, will not cause the system to overpressure, before starting any sequence. Goal: no surprises good unattended operation over 100’s or 1000’s ofsurprises, good unattended operation over 100 s or 1000 s of injections.
7. Avoid overtightening fittings/replace fitting to column periodically – use polyketone fittings for quick changes Goal: good connections nopolyketone fittings for quick changes. Goal: good connections, no extra volume, no overtightening of fittings.
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Mobile Phase and Sample Recommendations to Avoid High PressureAvoid High PressureIf the system has been sitting with buffer in it, flush the injector as well as the column. This prevents any bacterial growth in the injector from transferring to the column and plugging the fritthe column and plugging the frit.
Replace bottles of mobile phase buffer every 24 – 48 hours. Do not top off the bottle with more mobile phase, replace the buffer with a fresh bottle
Do not use a high buffer salt mobile phase (>50mM) in combination with high ACN concentrations due to possible precipitation.
Filter all aqueous buffers prior to use through a 0.2 um filter.
Use solvents that are high quality chromatography grade solvents (HPLC or MS grade).
Filter all samples with particulates through an appropriate 0 2um filterFilter all samples with particulates through an appropriate 0.2um filter. Particulates can clog the inlet frit on the column and cause high pressure and short column lifetime.
Install an in line filter to catch particulates before they get to the column
RR-Practical Considerations Agilent Restricted
5/6/2009
Install an in-line filter to catch particulates before they get to the column.
RRLC In-line Filter and Polyketone Fittings –Making RRLC and RRHT Columns Easy to UseMaking RRLC and RRHT Columns Easy to Use
Description Part number PorosityFrit diameter
Flow rate
Part number Replacement Frits
RRLC In lineRRLC In-line filter, 2.1 mm, max 600 bar
5067-1551 0.2 µm 2.1 mm <1 mL/min
5067-1555 (10/pk)
RRLC In-line filter, 3.0 & 4.6 5067 1553 0 2 µm 4 6 mm 1 - 5 5067-1562
Protect RRHT columns with efficient in-line filter with 0.2 µm pore size frits.
mm, max 600 bar
5067-1553 0.2 µm 4.6 mm mL/min (10/pk)
Easy, finger tighten column connection with Polyketone fittings up to 600 bar PN: 5042-8957 (10/pk)
Si l i t t d t d th lif ti f th l dSimple accessories can protect and extend the lifetime of the column and finger tight fittings are easy to use making column and tubing changes fast and leak free.
RR-Practical Considerations Agilent Restricted
5/6/2009
Sample and Injection Solvent Considerations – Are There Limits?There Limits?1. Injecting samples with particulates will plug the frit – filter
before use.
2. The injection solvent should be miscible with the mobile phase in the column at injection (gradient or isocratic).
3. Sample should be soluble with the initial mobile phase composition.
4 Injection solvent matched to initial mobile phase is ideal for4. Injection solvent matched to initial mobile phase is ideal for chromatographic efficiency and peak shape, but you can inject in DMSO
5. You can inject protein containing samples or other “dirty” samples, but you will need to clean up the column.
RR-Practical Considerations Agilent Restricted
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You Can Inject Dirty Samples – Customer Samples/ExamplesSamples/ExamplesExample 1:• RRHT SB-C18, 2.1 x 50mm, 1.8um 600 bar columnsRRHT SB C18, 2.1 x 50mm, 1.8um 600 bar columns• Protein sample dissolved in DMSO• Gradient mobile phase• High pressure LC
ObservationAft l 100 i j ti l i• After several 100 injections pressure on column increases
Question• Is the column still usable?Is the column still usable?
RR-Practical Considerations Agilent Restricted
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Example #1
Adsorbed material washing off – this column flushed with ACN:water
Pressure trace while column is equilibrating,the pressure increases are smalleras flow rate is increased because of materialthat has been washed off
Fl hi l d b d l t d lif ti
that has been washed off
RR-Practical Considerations Agilent Restricted
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Flushing column removes adsorbed sample, extends lifetime.
Example 1: Excellent Performance Achieved After “Cleaning” ColumnCleaning Column
Adsorbed material washing
Pressure change as adsorbed material eluted
goff
QC chromatogram
Pressure, efficiency return to initial levels after cleaning.C ti d i ibl Fl hi i d d t t d lif
RR-Practical Considerations Agilent Restricted
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Continued use is possible. Flushing is recommended to extend life.
Photo of Frits – No contamination of Frits
Frits are relatively clean indicating that a plugged frit with particulates was not the problem, but material from the sample adsorbing to the column was the issuecolumn was the issue. Few small black particles – typical of wear on any LC system
RR-Practical Considerations Agilent Restricted
5/6/2009
Example #2: Protein Precipitated Samples “Dirty Sample” Pressure vs. Injection #Sample Pressure vs. Injection #
150
Column: ZORBAX RRHT SB-C18
130
140Column: ZORBAX RRHT SB C182.1 x 30mm, 1.8 μm
120 End Press
Pressure Increase of only 8 bar over 600+ injections!!
100
110
900 100 200 300 400 500 600 700
Inj #Protein precipitated filtered – only sample preparation
RR-Practical Considerations Agilent Restricted
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Protein precipitated, filtered only sample preparationWith no particulates pressure increase is minimal.
Success #1: RRLC Gradients with the Agilent 1200 SL QA/QC Analysis: SB-C18 3.0 x 50 mm 1.8 um
GAT-XXX5708492-1
mAU3000
Customer Sample - drug intermediate QA-sample
1 Blanks1 T t l
2000
2500GAT-XXX5708492-1
mAU
2500
1 Test-sample (ref. of main comp. and imp.)
2 Std.-samples (reference of main comp.)
1 Sample2 Std.-samples
70 C, pc cooling 21 C flow = 2.8 ml/min A= (2mM Phos, pH=2.0), B=ACN50 90%B in 3 0min 90%B
1000
1500
XXX6087077
1500
2000S d sa p es
1 Sample1 Std. Sample
Original method was
50-90%B in 3.0min, 90%B 1.5minanalysis-time = 4.5 minpost-time = 1.0minDAD SL: 214 & 266nm
min0 5 10 15 20 25 30
0
500XXX6087077
XXX6087077500
1000Original method was 35 min run time= 5 hours 15 min = half a day!!!
30
min0.5 1 1.5 2 2.5 3 3.5 4.0
0
RRLC Method:5.5 min run time
49 i til b t h l !!!RR-Practical Considerations
Agilent Restricted5/6/2009
= 49 min until batch release!!!
1200 RRLC – Achieve Maximum Results with RRHT Columns for High Speed and High Resolution LC
Increased linear flow rates for higher analysis speedHigher Column and System Temperature: 80°C => 100°C
C & S
Columns for High Speed and High Resolution LC
Higher Allowed Column & System Pressure: 400bar => 600barMinimized column pressure: – 25%Minimized system pressure: – 30% at 1ml/minMaintained high flow rates: 0 05 5ml/minMaintained high flow rates: 0.05 – 5ml/min
High Method FlexibilitySupport of narrow (2.1mm) and analytical (3.0 and 4.6mm)
columnsSupport of RRLC and conventional HPLC on one unitBroad Range of >100 RRHT 1.8um ColumnsSame Chemistry for 1.8, 3.5, 5 and 7um particle columns
Fast, easy, secure method transferIncreased efficiency for higher resolution/peak capacity
Longer columns: 50mm => 150mm
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Conclusions
The key advantages or Rapid Resolution HT (1.8um) columns are improved speed and resolution.
Th “li it ti ” f RRHT l f d ithThe “limitations” of RRHT columns are very few and with a suitable focus on reducing particulates excellent results can be obtained.
All normal mobile phases and normal conditions can be used on RRHT columns.
RRLC instruments provide the ideal tool for achieving high resolution and high throughput results with RRHT columns.
RR-Practical Considerations Agilent Restricted
5/6/2009
Agilent LC Columns and Agilent J&W GC Columns Scientific Technical SupportColumns Scientific Technical Support
800-227-9770 (phone: US & Canada)*
302-993-5304 (phone)
For LC columnsFor LC columns
Select option 4, then option 2
For GC Columns
* Select option 4, then option 1.
www.agilent.com/chem
RR-Practical Considerations Agilent Restricted
5/6/2009
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