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Page 1
Making Sub-2Micron Particle
Technology Easy
LC Columns and Consumables
John PalmerChromatography SpecialistOctober 12, 2007
Page 2
Bonded phase
Bonded phase
• Differential partitioning of the components into the stationary and mobile phases.
• Separation controlled by chemical interaction of mobile phase/sample/bonded phase
Mobile phase
The Separation Process
Page 3
Separation Mechanism
• As Bands Travel the Length of the Column the Respective Distance(ΔL ) Increases
• As Bands Move Through the Column Dispersion (while in the Bonded Phase and Mobile Phase) Causes the Band Width to Increase
• Choosing a High Efficiency Short Column Minimizes On-Column Dispersion and Improves Resolution
W1 W2ΔLL
Page 4
Longitudinal diffusion
Smaller Particles Improve HETP by Minimizing Dispersion Effects
Plat
e H
eigh
t H
Linear Velocity u
Eddy Diffusion
Sum Curve: van-Deemter
Resistance to Mass Transfer
H = A + B/u + C uLarge Particle
SmallParticle
The smaller the plate height, the higher the plate number and the greater the chromatographic resolution
u opt
H min
Page 5
Smaller Particles Maintain Efficiency Over Wider Flow Rate Ranges
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Column: ZORBAX Eclipse XDB-C18 Dimensions: 4.6 x 50/30mmEluent: 85:15 ACN:WaterFlow Rates: 0.05 – 5.0 mL/minTemp: 20°CSample: 1.0μL Octanophenone in
Eluent
Volumetric Flow Rate (mL/min)
HET
P (c
m)
5.0μm3.5μm1.8μm
H = A + B/u + Cu
Smaller particle sizes yield flatter curves, minima shift to higher flow rates
Page 6
ZORBAX RRHT Columns Provide the Efficiency of Longer Columns for More ProductivityColumnLength(mm)
Resolving Power
N(5 µm)
Resolving Power
N(3.5 µm)
ResolvingPower
N(1.8 µm)
Typical Pressure
Bar (1.8 µm)
150 12,500 21,000 32,500 >420
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
AnalysisTime
PeakVolume
SolventUsage
Analysis Time*
-33%
-50%
-67%
-80%
-90%
* Reduction in analysis time compared to 150 mm column• pressure determined with 60:40 MeOH/water, 1ml/min, 4.6mm ID
Page 7
Pressure Equation Defines Physical Causes�
ΔP = ηFLK0πr2dp
2
viscosity flow length
specificPermeability
columnradius
particlediameter
The particle size term is absolute, and does not consider the particle size distribution
Page 8
Ultrafast HPLC (UFLC)First Publication High Resolution/Speed LC with standard Agilent 1100 LC and Zorbax 1.8um particle
•J. Chrom Sci., 38, Dec 2000, pp 535-544
• author J. J. Kirkland
•Review article with 38 references
•Binary 1100 pump, using 1.8 um 4.6 x 30 mm columns
•Appl’s demonstrating 15 and 24 sec separations
Page 9
How Does ZORBAX RRHT EBT Manage Pressure for Universal Application?
1. Investigate the effect of Particle Size Distribution (PSD) on performance to develop new packing bed process
2. Take extra steps to minimize fines and therefore lower column backpressure.
3. INTENTIONALLY leave a small, controlled fraction of slightly larger particles in the commercial product because: Slightly broader PSD packings resulted in LITTLE loss of column efficiency compared to the narrow PSD packings.Narrower PSD packings resulted in columns with MUCH higher back pressures. The narrower PSD packing produced a backpressure that was 25% higher than the packing with an optimized PSD
Page 10
Actual Example of Particle Size Distribution on ZORBAX 1.8um Particles
Number Average Plot of ZORBAX 1.8 um material
Differential Number
6543210
500
1000
1500
2000
2500
3000
3500
Particle Diameter (μm)
Num
ber
Number StatisticsMean: 1.740 umMedian: 1.727 umMode: 1.732 um95% Conf. Limits: 1.739 – 1.742 umSD.: 0.271 um
%> 10 90um 2.04 1.47
Small number of “off median” particles visible in number average plot
Page 11
Van Deemter Curves for Narrow vs. Engineered Particle Size Distribution 1.8μm SB-C18
0
1
2
3
4
5
6
7
8
9
10
0 2 4 6 8 10 12 14 16 18 20Reduced Linear Velocity (ν)
Red
uced
Pla
te H
eigh
t (h)
Production 1.8μm – Engineered PSD
Narrow 1.8μm – Narrow PSD
7793 Plates @
1.6 mL/min(-3.4%)
8065 Plates @
2.0 mL/min
Columns: ZORBAX SB-C18Dimensions: 4.6 x 30mm, 1.8 μmEluent: 85:15 ACN:WaterFlow Rates: 0.05 – 5.0 mL/minTemp: 20°CSample: 1.0μL Octanophenone in
EluentDetection: UV 245nm
h vs. υ Fit CoefficientsProduction PSD Narrow
PSDVD A: 0.795 0.760
B: 3.97 4.26C: 0.085 0.077r2: 0.9994 0.9998
Page 12
Particle Size Distribution – Agilent vs. WatersAgilent has a tighter, narrow PSD
Packing from a new Waters Acquity 2.1 x 100mm, 1.7um column
Page 13
HETP vs. Volumetric Flow RateSmall Particle Columns including Monolith
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Dimensions: 4.6 x 50/30/20mmEluent: 85:15 ACN:WaterFlow Rates: 0.05 – 5.0 mL/minTemp: 20°CSample: 1.0μL Octanophenone in
Eluent
Volumetric Flow Rate (mL/min)
(cm
/pla
te)
HET
P
ZORBAX 5.0μmZORBAX 3.5μmVendor A 2.5μmVendor B 2.0μmZORBAX 1.8μmMonolith
Page 14
Agilent RRHT Columns Show No Selectivity Differences with Changes in Particle Sizes Under Same Conditions
Column: 4.6 x 50mm Eclipse XDB-C18 Mobile Phase A= pH 4.5 Na Acetate, B=MeOH (60:40)Flow Rate =1 mL/min Inj. Vol: 2 ul Flowcell: 3uL, 2 mm flow path
1. Caffeine (I.S.)2. Allobarbital3. Phenobarbital4. Butabarbital5. Hexobarbital
min1 2 3 4 5 6
min1 2 3 4 5 6
min1 2 3 4 5 6
5 µm
1.8 µm
3.5 µm t0=0.44 minα 3,2=1.20
t0=0.44 minα 3,2=1.20
t0=0.46 minα 3,2=1.22
α 4,3=1.97
α 4,3=1.94
α 4,3=1.97
α 5,4=1.13
α 5,4=1.16
α 5,4=1.14
Page 15
Why Worry About pH?pH, pKa and Weak Acids
At pH 4.2 – the sample exists as benzoic acid and the benzoate ion in a ratio of 1:1. Peak shape can be poorAt pH 5.2 – 91% of the sample exists as the benzoate ion. RP retention decreases.
At pH 3.2 – 91% of the sample exists as benzoic acid. RP retention increases.
RCOOH RCOO- + H+
Ka = 6.4 x 10-5
pKa = 4.2
Ka =[RCOO-][H+]
[RCOOH]
+ H+
COOH COO_
Page 16
Why Worry About pH?pH, pKa and Weak Bases
At pH 9 – the sample exists as protonated and unprotonated diphenhydramine in a ratio of 1:1. Peak shape can be poor.At pH 10 – 91% of the sample exists as unprotonated diphenhydramine.At pH 8 – 91% of the sample exists as protonated diphenhydramine.
Ka =[R3N][H+][R3NH+]
Ka = 1 x 10-9
pKa = 9
R3NH+ R3N + H+
CHOCH CH N2 2
CH3
CH3
∅
∅
+H
CH3
CH3
∅
∅
CHOCH CH N2 2 + H+
Page 17
pH vs. Selectivity for Acids and Bases
5
SCD
++
2 +
1
1.5
1.0
0.5
0.0
-0.5
log k«
3 4 5 6 7 8 pH
A B C
2
4
5
3
3 5 7 9ELUENT pH
40
30
20
10
10Re
tentio
n
+++
3
1 7,12 - OC
UDCSOC4
6
C12-OC
J.C. 268(1983) 1
J.C. 111(1975) 149
Column: Nucleosil-C18Mobile Phase: 45% ACN/55% phosphate bufferSample: Bile Acids
Column: μBondapak-C18Mobile Phase: 60% 25 mM phosphate buffer
40% Methanol1. Salicylic acid2. Phenobarbital3. Phenacetin4. Nicotine5. Methamphetamine
• Retention and selectivity can change dramatically when pH is changed.
Page 18
0 5
1
2,3
4
5
Time (min)
7
6
0 5 10
1
2
3
4
Time (min)
6
5
7
High pH Improves Retention and Resolution of Antihistamines
Peak Shape and Retention of this sample of basic compounds improves at high pH. Why?
Column: ZORBAX Extend-C18, 4.6 x 150 mm, 5 μ m Mobile Phase: See Below Flow Rate: 1.0 mL/min Temperature: RT Detection: UV 254 nm Sample: 1. Maleate 2. Scopolamine 3. Pseudoephedrine 4. Doxylamine 5. Chlorpheniramine 6. Triprolidine 7. Diphenhydramine
pH 730% 20 mM Na2HPO470% MeOH
pH 1130% 20 mM TEA70% MeOH
tR = 8.5 tR = 11.4
Page 19
Resolution Equation Which Parameter Has Most Effect on Resolution?
Rs =N
4
k'
k'+1
(α-1)
000990P2.PPT
Plates Selectivity Retention
••α
For the Answer Let’s Take Look at the Next Slide!
Page 20
Resolution as a Function of Selectivity, Column Efficiency, or Retention
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Res
olut
ion
Increase NIncrease AlphaIncrease k'
Plates: 5000 10000 15000 20000 2500Alpha: 1.10 1.35 1.60 1.85 2.1k’: 2.0 4.5 7.0 9.5 12.0
Selectivity Impacts Resolution Most• Change bonded phase • Change mobile phase
Rs = N½/4 • (α-1)/α • k’/(k’+1)
Page 21
ZORBAX RRHT Bonded Phase Choices
ZORBAX Eclipse XDB – first choice for method development, general purpose bonded phase, for most applications, use up to 60°C
ZORBAX Eclipse PLUS– excellent choice for method development, optimized for basic compounds but applicable for neutrals and acids, use up to 60°C
ZORBAX StableBond – good for low pH and high temperature applications, for those who want to raise temperature for faster analysis
• SB-C18 – use up to 90°C and down to pH <1 (high temp only at low pH)• SB-C8 – use up to 80°C and down to pH 1• SB-CN – use up to 80°C at pH 2• Note: Acquity can only go up to 75°C
ZORBAX Extend-C18 – good at high pH – up to pH 11.5 at ambient temperature
Match Method pH and Column ChoiceChoose the Best Bonded-Phase for Each pH Range
StableBond, pH 1-61. Uses bulky silanes2. Non-endcapped
Eclipse Plus and XDB, pH 2-91. eXtra Densely Bondeddimethylalkylsilanes2. proprietary double-endcapping
*
Bonus-RP, pH 2-81. polar alkyl phase2. triple endcapped3. uses bulky silanes
Extend-C18, pH 2-11.51. unique bidentate structure2. double endcapped*
O
R
R
Si
OH
Si
O
Si
R
R
R
O
OH
R
R1
R1
R1
SiO
SiO
Silica Support
C18C18
CH3
CH3
CH3
CH3
CH3
CH3
O
SiO
O
O
O
Si
Si
Si
CH3
CH3
CH3
CH3CH3
CH3
Si
O R
Si
Si
O
Si
O R
R1
CH3
CH3
CH3
CH3
R
R1
CH3
R1
R1
R1
CH3
R1
Si
Si
PG
PG
PG
Page 23
Eclipse Plus C18 – Best Peak Shape for Bases without Tailing – Standard Test Mix 1
Eclipse Plus C18
Ace 5 C18
Discovery HS C18
Gemini C18
Luna C18(2)
SunFire C18
XTerra MS C18
XBridge C18
min0 0.5 1 1.5 2 2.5 3 3.5
1. Pyridine (Base) 2. Phenol (Acid) Mobile Phase: 60/40 Water/ACN
12
1 2
1 2
12
12
1
1
2
2
1 2
Page 24
Start Method Development with RRHT Columns: Different ZORBAX RRHT C18 Bonded Phases for Max Selectivity
Eclipse Plus C18
Eclipse XDB-C18
Extend-C18
StableBondSB-C18
Mobile phase: (69:31) ACN: waterFlow 1.5 mL/min.Temp: 30 °CDetector: Single Quad ESI positive mode scan Columns: RRHT 4.6 x 50 mm 1.8 um
1 23
4
Sample:1. anandamide (AEA)2. Palmitoylethanolamide (PEA)3. 2-arachinoylglycerol (2-AG)4. Oleoylethanolamide (OEA)
1 2 3 4 5
1 23
4
1 2 3 4 5
1 2,3 4
1 2 3 4 5
1 234
min1 2 3 4 5
1st choiceBest Resolution& Peak Shape
2nd choiceGood alternate selectivity due to non-endcapped
3rd choiceGood efficiency & peak shapeResolution could be achieved
4th choiceResolution not likely,Other choices better, for this separation.
Multiple bonded phases for most effective method development.
Page 25
More RRHT Bonded Phase Choices Allow for Optimized Method Development
Conditions: Columns: RRHT 4.6 x 50mm, 1.8um Mobile Phase: Acetonitrile (40%):Buffer 60% pH=2.4 25mM NaH2PO4Flow Rate: 1.85 mL/min Detection: DAD, 254 nm LC: Agilent 1200 RR LC Sample: As listed, 1 uL injection
Change in Elution Order Peaks 3,4 & 5
Sample:1. Tolmetin2. Naproxen3. Diflunisal4. Ibuprofen5. DiclofenacFor SB-Phenyl
SB-Phenyl 1.8um
SB-C8 1.8um
SB-C18 1.8um
SB-CN 1.8um
min0 1 2 3 4 5
mAU
0
50
100
150
200
250
300
350
400
450
5 Choices in SB family can be used for method optimizationRRHT SB-Phenyl best choice - more resolution in half the time!
Best Choice!
2X the needed timeNot the best choice!
Page 26
The Complete List of RRHT 600 bar Columns As of April 9, 2007
Dimensions Eclipse Plus C18 Eclipse Plus C8Eclipse
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 959994-3023.0 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 728700-9022.1 x 50 959741-902 959741-906 927700-902 927700-906 727700-9022.1 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 827975-9014.6 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)http://www.chem.agilent.com/Scripts/Generic.ASP?lPage=11424&indcol=N&prodcol=N (P/N List)
2 Recommended Starting Choices:• 959941-902• 959741-902They are Eclipse Plus C18 Columns.
Page 27
Equivalent Selectivity and Improved Efficiency ZORBAX Eclipse Plus Substituted for Original Column
min0 2 4 6 8 10 12
min0 2 4 6 8 10 12
Waters X-Bridge C18, 4.6 x 150 mm, 5 um
ZORBAX Eclipse Plus C18, 4.6 x 150 mm, 5 um
N=41000TF=1.13
N=14000TF=1.25
N=60000TF=1.00
N=23000TF=1.01
Gradient: 10 to 30% B/15 min. A: 0.1% Formic Acid, B: 0.1% FA in ACN Flow: 1 mL/min. Temperature 40°CElution order: sulfanilaminde, sulfadiazine, sulfathathiazole, sulfamerazine, sulfamethazine, sulfamethoxazole
Page 28
Let Us Take A Break!
Page 29
RRLC Instrument Considerations:Can the HPLC in your lab today handle RRLC ?
Gradient delay volume
Extra-column volume
Data rate
•Things to consider on your system:•Gradient delay volume – effects column re-equilibration and gradient profile
•Extra-column volume – effects peak dispersion and peak width for Isocratic separations
•Data Rate – match to expected peak widths-If rate is too slow sensitivity and peak detection suffer in Iso and Gradient separations
Page 30
Binary Pump SL• Configurable delay volume (120µl and 600-800µl) for
uncompromised support of narrow and std bore columns (2.1 – 4.6mm ID)
– Standard delay volume (600 – 800 µl) for uncompromisedsupport of std bore RRLC and HPLC on one configuration
– Low delay volume (120 µl) for uncompromised support of narrow LC and LC/MS (both RRLC and HPLC)
• 0.05 – 5 ml/min flow rate range for highest speed on narrow and standard bore RRHT columns (full P range)
• 600 bar pressure for higher speed and resolution in RRLC• Highest RT precision and lowest mixing noise by new
piston control algorithms (EDC – Electronic Damping Control)*
*patents filed
1200 RRLC System Benefits of Key Modules Features
Page 31
1200 Series Binary Pump SL – Configurable Delay Volume
Low delay volume
120 µl
Disconnect only here!
BA
Pressure Sensor
PurgeValve
600bar Damper
400µl Mixer
Pressure-Sensor
Purge Valve
Mixing-T
Pressure-Sensor
Purge Valve
Mixing-T
Damper
400µl Mixer
Std delay volume
600-800 µlBA
600bar Damper
400µl Mixer
Pressure Sensor
Purge Valve
Disconnect only here!
Flow Path
Page 32
min0 0.2 0.4 0.6 0.8 1
mAU
0
100
200
300
400
500
600
700
0.20
9
0.37
0
0.50
5
0.62
00.
664
0.72
5
0.82
1
0.90
8
0.98
6
1200 Rapid Resolution
Suppler B
Column RRHT, SB-C18,
2.1 x 50mm, 1.8um
A BEH,
2.1 x 50mm,1.7umAnal. Time 0.949 min 0.914 min
Resolution 3.69 3.50
Precision 0.025 – 0.094 %RSD 0.18 – 0.30 %RSD
1200 Series Rapid Resolution System - efficient use of pressure
Pressure 440 bar 700 bar (+60%)
Sample: Phenones Test MixFlow Rate: 1 ml/minGradient: 35-95% ACN in 0.9
minTemperature: 50°Injection volume: 1 µlInjection Technique: ADVR,
OI, MCOWL: 245 nmData Rate: 80 Hz
Better performance at dramatically lower pressure than other U-HPLC designs
Page 33
1200 RRLC and RRHT Column FlexibilityConventional to High Speed HPLC
Conditions:A=pH 4.5 Na Acetate, B= Methanol (60:40)1.0mL/minFlow cell: Micro/High Pressure 6mm, 1.7µLDetection: UV 254 nmInjection Volume: 2 uL
min0 1 2 3 4 5 6 7
Rapid Resolution HTEclipse Plus C18
4.6 x 50 mm, 1.8µm
Rapid ResolutionEclipse Plus C18
4.6 x 150 mm, 3.5 µm
Eclipse Plus C184.6 x 250 mm, 5 µm
min0 5 10 15 20 25 30
min2 4 6 8 10 12 14 16 18
Sample: Phenobarbital 1. Caffeine 2. Allobarbital 3. Phenobarbital 4. Butabarbital 5. Hexobarbital
Page 34
main comp. • Zorbax SB C18 2.1mm x 150mm, 1.8µm• 20-95%ACN (0.1%TFA) in 14min, 2min hold,
5min 20%ACN, 55°C, 0.5ml/min• total runtime 21 min
1
2
3
45
6
78
Customer Sample 1
1200 Rapid Resolution System – PerformanceIncreased Resolution in Drug Development Impurity Testing
• Acquity BEH C18 2.1x100mm,• 5-80% ACN (50mM NaClO4/0.1%H3PO4) in
12min, in 0.5min to 5% ACN, 8.5min 5% ACN, 0.5ml/min, 40°C,
• total runtime 21 min
1 2 3
4
56
main comp.
• Two more baseline separated impurities • Higher sensitivity, lower noise• Same runtime
Page 35
Stepwise Scale-up to Rapid Resolution LCFrom 1100 to 1200 RRLC in two steps – Example 1
Actual: 1100
Quat System
> 5 min> 6 min> 3 sec
5 - 12,0004.6 mm50 mm
0.2 - 10ml/min80 C
400bar
ALS
TCC
VWD/MWD/DAD
Quat Pump
Degasser
Step 1: 1100/1200
„Bin SL“ System
h-ALS SL
TCC
VWD/MWD/DAD
Bin Pump SL
µ-Degasser
> 1.5min> 2min
> 1.5 sec5 - 30,000
2.1 - 4.6 mm50 - 150 mm
0.05 - 5 ml/min80 C
600 bar
Analysis TimeCycle timesPeak Width
NColumn ID
Column Length Flow rates
TemperaturePressure
Step 2: 1200
Rapid Resolution System
h-ALS SL
TCC SL
DAD SL
Bin Pump SL
u-Degasser
> 0.2min> 0.4min> 0.2 sec
5 - 60,0002.1 - 4.6 mm20 - 150mm
0.05 – 5 ml/min100 C
600bar
+ Speed+ Resolution+ Sensitivity
+ MS-Robustness+ Data Security & Traceability
+ Qualification (Degasser, ACE)+ Compatible with conv. HPLC
+ Resolution+ Speed
+ MS-Compatibility+ Solvent Saving
+ Compatible with conv. HPLC
Preserve your existing 1100 LC module investment:
Page 36
Agilent HPLC System Flexibility
• Agilent LC systems are designed for flexible use with a wide range of flow and pressure capabilities
– Many of the New 1200 Modules (i.e. 80 Hz DAD) are compatible with older 1100 systems
– 1200 RRLC systems can run current, legacy and new high performance methods
– Engineered Column Technology Allows Higher Performance Separations with:
– Existing 1100 System– Enhanced 1100 System – Higher Pressure, Enhanced Flow Path 1200SL System
Separation needs will determine your choice!
Page 37
New RRHT-1100 Upgrade Kits Make Conversion of 1100 for use of RRHT Columns
5188-5323 4.6 mm RR HT 1100 Fast LC Kit – VWD- 0.17 mm capillaries, 80 ul mixer, 5 ul semi-micro VWD cell- two 4.6 x 50 mm Zorbax 1.8 um columns
5188- 5324 4.6 mm RR HT 1100 Fast LC Kit – DAD- 0.17 mm capillaries, 80 ul mixer, 5 ul semi-micro DAD cell- two 4.6 x 50 mm Zorbax 1.8 um columns
5188-5328 2.1 mm RR HT 1100 Fast LC Kit – MS/DAD- 0.12 mm capillaries, 80 ul mixer, 1.7 ul micro DAD cell- 0.12 mm std ALS and WP ALS needle seats- two 2.1 x 50 mm Zorbax 1.8 um columns
Page 38
Possible Instrument Configurations for Cost Effective Fast and Ultra-Fast HPLC
* Pieces to upgrade, in kits
High pressureGradient pump
Std or WellPlate sampler
Diode Array Detector
Standard assembly without standard mixer
0.12 x 400 mm capillary
DAD equipped with a 1.7 μL flow cell
Mass Spectrometer
0.13 x XX mm PEEK Capillary
High pressure Gradient pump
Std or WellPlate sampler
Rapid ResolutionHT Column
Diode Arraydetector
Waste
Standard assembly without standard mixer and with 80 μL mixer
0.17 x 400 mm capillary
0.17 x 150 mm capillary
0.17 x 105 mm capillary
DAD equipped with a 5uL or 1.7 μL flow cell
3 μL heat exchanger
Thermostatted Column
compartment
4.6 mm ID columns 2.1 mm ID columns
Cell Inlet Capillary
Cell Outlet Capillary
Rapid ResolutionHT Column
0.12 x 150 mm capillary
0.12 x 105 mm capillary
3 μL heat exchanger
Thermostatted Column
compartment
DAD equipped with a 500 nL flow cell
Cell Inlet Capillary
Cell Outlet Capillary 0.125 x 80 mm capillary
0.125 x 220 mm capillary
Page 39
The Agilent 1200 LC SystemsOne Comprehensive Series of Solutions for your Applications
Application 1200 RRTM LC 1100/1200 Binary
1100/1200 Quaternary
Ultra-fastcolumn length 15 – 50mm
YES NODelay volume too large
NODelay volume too large
RRLC – Narrow
(2.1)High-resolutioncolumn length 100 – 150mm
YES NODelay volume too large, pressure limit too low
NODelay volume too large, pressure limit too low
Ultra-fastcolumn length 15 – 50mm
YES YES YESModifications
recommended to reduce delay volume
RRLC – Std
(3.0, 4.6)
High-resolutioncolumn length 100 – 150mm
YES NOPressure limit too low
NOPressure limit too low
HPLC – Narrow bore (2.1) YES YESModifications
recommended to reduce delay volume
NODelay volume too large
HPLC – Standard bore (3.0, 4.6) YES YES YES
Page 40
Transferring a Method- Binary to QuaternaryThe Proof that a binary to quaternary works!
min0.5 1 1.5 2 2.5
mAU
0
100
200
300
400
500
600
DAD1 A, Sig=210,4 Ref=off (FASTLC\02090510.D)
1.5
45 1
.606
1.8µm 4.6x50mm Binary Pump
1.8µm 4.6x50mmQuat Pump
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40
Com
poun
d "F
" - 1
.434
Com
poun
d "G
" - 1
.493
1.65
41.
709
Com
poun
d "D
" - 1
.736
Run time 2.3 minutes
Run time 1.8 minutes
Run time differs by 0.5 minutes due to larger delay volume in quaternary pump.But separation acceptable to the customer.Making use of available instruments with some limitations.
Page 41
Higher Temperature as an Aid to Faster Operation
Higher Temperature:
Temperature should always be considered as a parameter during speed optimization
Provides more rapid mass transfer:• Improves Efficiency – enhances resolution• Decreases analysis time – faster separations with no loss in resolution
Decreases Mobile Phase Viscosity• Lowers backpressure – allows for higher flow rates, faster separations,
greater efficiency and use of sub 2-micron columns
Can change selectivity – optimize resolution
Page 42
High Temperature for Improved Selectivity – What Column Compartment is Required?
20°C
60°C
40°C
30°C
90°C
Salicylic acid
min0 1 2
0 1 2 345
0 1 2 345
0 1 2 345
0 1 2 345
3 4
Salicylic acidCoelution
Salicylic acid
Salicylic acid
Salicylic acid
Column: RRHT SB-C184.6 x 50mm, 1.8um
Increased T Decreases Run Time and Sharpens Peaks Improving Resolution
Page 43
1200 Series TCC SL Low volume Pre-column Heater and Post-column Cooler
• L-shaped pre-column heater • Volume: 1.6 µl• Mounted on carrier
• U-shaped Post-column cooler • Volume: 1.5 ul• Mounted on Carrier
Holesto attachcarrier
Choice of Valves
Plumb multiple columns – have it your way !
Page 44
The New 1200 Thermostatted Column Compartment SLPost-column coolingNoise reduction at high flow rates and high temperatures
0.685 - Anthracene
min0.5 1 1.5 2 2.5
mAU
-0.05
0
0.05
0.1
0.15
0.2
0.25
• Column Temperature: 80°C • Detector-in Temperature: 50 °C• Noise = 90 uAU• S/N = 3.3
• Column Temperature: 80°C • Detector-in Temperature: 24 °C• Noise = 30 uAU• S/N = 10• No Peak Broadening
Column: 4.6 x 50mm, 1.8um
Flow Rate: 4ml/min
Detector celltemperature
Detector celltemperature
Page 45
Data Rate
Peak Width
Resolution Peak Capacity
80 Hz 0.300 2.25 60
40 Hz 0.329 2.05 55
20 Hz 0.416 1.71 45
10 Hz 0.666 1.17 29
5 Hz 1.236 0.67 16
min0.1 0.2 0.3 0.4 0.50
80Hz
PW=0.30sec
40Hz
PW=0.33sec
20Hz
PW=0.42sec
10HzPW=0.67sec
5HzPW=1.24sec
Sample: Phenones Test MixColumn: Zorbax SB-C18, 4.6x30, 1.8umGradient:: 50-100%ACN in 0.3minFlow Rate: 5ml/min
80Hz versus 10Hz (20Hz) Data Rate• Peak Width: – 55% (– 30%)• Resolution: + 90% (+ 30%)• Peak Capacity: + 120% (+ 40%)• App. Column Eff.: + 260% (+ 70%)
High Speed LC with RRLC and RRHT ColumnsMatch Detector Speed to Peak Width for Optimum Detection
Page 46
Chromatographic Results with Second Source Lamp at 214 nm Wavelength - Small Peaks
Lamp fromLamp from AgilentAgilentTechnologiesTechnologies
Lamp from Second SourceLamp from Second Source
Peak 1Peak 1 S/N = 150S/N = 150
Peak 2Peak 2 S/N = 400S/N = 400
Peak 3Peak 3 S/N = 300S/N = 300
Peak 1Peak 1 S/N = 15S/N = 15
Peak 2Peak 2 S/N = 50S/N = 50
Peak 3Peak 3 S/N = 50S/N = 50
Page 47
Narrow Peaks Require Faster MS Scan Speeds
min0.1 0.2 0.3 0.4 0.5 0.6 0.7
50000
100000
150000
200000
MSD1 TIC, MS File (C:\CHEM32\1\DATA\FAST SCAN 7_05\SULFA1000034.D) MM-ES+APCI, Fast Scan, Frag: 150
0.10
2
0.16
7
0.43
2
0.48
1
PW = 0.8 sec
RRHT SB-C18 2.1 x 50 x 1.8 micron column, 5% MeOH w 1 mM ammonium acetate to 50% in 1 minute
Data acquired with 6140 MS – fast enough for RRHT columns.
Page 48
Increased Isocratic HPLC Speed with 1.8u Particles
1. Reduce Column Length and Particle Size
2. Maintain Flow Rate
3. If Resolution and Pressure is Within Goal, Increase Flow Rate
4. Approaching Pressure Limit of Instrument ? Increase Temperature to Lower Viscosity and Lower Pressure
5. Increase Flow Rate
6. Approaching Flow Rate Limit of Instrument ? Decrease Diameter Column and Reduce Flow Rate Proportionately
7. Repeat Steps 3, 4 and 5 until Speed Goal Achieved ISOCRATIC ELUTION
Page 49
Faster AnalysisShorter Column, Smaller Particle, Same Flow
Original methodZORBAX LC column Extend-C184.6 x 150 mm, 5 μm3 μL inj.
min0 2 4 6 8 10
mAU
0
50
100
150
200
250
min0 2 4 6 8 10
mAU
-20
0
20
40
60
80
100
120
Mobile phase: (70:30) MeOH: 50 mM pyrrolidine buffer Flow = 1.0 mL/min, Temp. : ambient
ZORBAX RRHT column Extend C184.6 x 50 mm, 1.8 μm1 μL inj.
2
2 3
5
4
516
614
3
3x faster
Page 50
Simplified Method Transfer for Increased Gradient Speed
1. Shorten the Column Length2. Adjust Gradient Time by same Factor 3. Maintain Flow Rate4. If Resolution and Pressure is Within Goal,
Increase Flow Rate and Lower Gradient Time5. Approaching Pressure Limit of Instrument ?
Increase Temperature to Lower Viscosity and Lower Pressure
6. Increase Flow Rate and Lower Gradient Time7. Approaching Flow Rate Limit of Instrument?
Decrease Diameter Column and Reduce Flow Rate Proportionately
8. Repeat Steps 4, 5, 6 and 7 until Speed Goal Achieved
GRADIENT ELUTION
0 10 20 30 40
Relationship of k* and Key Gradient Parameters
Time (min)
100% B
100% B
100% B
100% B
tg= 40
tg= 20
tg= 10
tg= 5
1/k* = gradient steepness = b
tg F
S Δ%B Vm
k* ∝
ΔΦ = change in volume fraction of B solvent
S = constantF = flow rate (mL/min.)tg = gradient time (min.)Vm = column void volume (mL)
0% B
0% B
0% B
0% B
Page 52
1
1
33
5
5
22
4
4
Time (min) Time (min) Time (min)0.0 2.5 5.0 7.5 10.0 12.5 15 0.0 2.5 5.0 7.5 0.0 2.5 5.0
1
3
5
2
4
Length = 150 mm, 5 μmtG = 18 minTotal Time ~ 30min
A. B. C.
Length = 75 mm, 3.5 μmtG = 9 minTotal Time ~ 15 min
Length = 50 mm, 3.5 μmtG = 6 minTotal Time ~ 8min
Column Diameter: 4.6 mm Mobile Phase: A: 25 mM Na2HPO4, pH 3 B: MeOH Gradient: 45 – 90% B in tG min Flow Rate: 1.0 mL/min Temperature: 35°C Sample: Cardiac Drugs 1. Diltiazem 2. Dipyridamole 3. Nifedipine 4.
Lidoflazine 5. Flunarizine
Reducing Column Length Reduces Run TimeGradient Time ∝ Column Length
Page 53
Rapid Resolution LCUltra 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 μm
Mobile Phase:A: 55% 25 mM Na2HPO4, pH 3B: 45% MeOH
Gradient: 42 – 90% B in 2 minFlow Rate: 3 mL/minTemperature:35°CSample: Cardiac Drugs
1. Diltiazem2. Dipyridamole3. Nifedipine4. Lidoflazine5. Flunarizine480 Samples/day
Run Time 1.8 minEquilibration 1 minTimeTotal Analysis 2.8 minTime
Page 54
F= 1.20ml/minT = 40°CAnalysis Time = 11minSolvent Cons. = 13.2ml
High Resolution:4.6mm x 150mm 5.0µm
min0 2 4 6 8 10 12
F = 4.80ml/minT = 40°CAnalysis Time = 1.05minSolvent Cons. = 5.1ml
High Speed:4.6mm x 50mm 5.0µm
min0 0.2 0.4 0.6 0.8 1
F= 1.00ml/minT = 40°CAnalysis Time = 1.1minSolvent Cons. = 1.1ml
High Speed & Resolution:2.1mm x 50mm 1.8µm
min0.2 0.4 0.6 0.8 10
Agilent 1200 Rapid Resolution System – SpeedConventional LC → UFLC → RRLC
Max Speed at T = 95oC2.1mm x 50mm 1.8um
F= 2.40ml/minT = 95°CAnalysis Time: 0.4minSolvent Cons. = 1.0ml
PWHH = 197msec
min0.2 0.4 0.6 0.8 10
> 20x faster !
Page 55
Rapid Resolution Compendium CD-ROM
(Please Ask For A Copy – Pub No 5989-5130EN)
Tools
• Application Examples
• Technology Overview
• Rapid Resolution in The Scientific and Trade Press and Conference Posters
• System Configurator (guide to reduce dead volume and extra column effects)
• Method Translator Program
Page 56
Mini-Demo Method Translator
Agilent HPLC Method TranslatorA Simple to Use Tool to Move Methods to RRHT
Page 57
Why Filter the Sample? Extreme Performance Requires Better Sample “Hygiene”
• Prevents blocking of capillaries, frits, and the column inlet
• Results in less wear and tear on the critical moving parts of injection valves
• Results in less downtime of the instrument for repairs
• Produces improved analytical results by removing potentially interfering contamination
Page 58
Choice of Membranes
• Regenerated Cellulose (RC)• Universal hydrophilic membrane, compatible with most
solvents - aqueous and organic• High purity, extremely low extractables and lowest protein
binding• PTFE
• Good chemical compatibility with aqueous and organic solvents as well as for acids and bases
• Hydrophobic• Nylon
• Universal filter for aqueous and organic solutions• Hydrophilic
• Cellulose Nitrate• Compatible with many, but not all, aqueous or non-
aqueous solvents• Most commonly used as a pre-filter
• Cellulose Acetate (CA)• Only compatible with aqueous solvents• Used for proteins and protein-related samples
Regenerated Cellulose
Nylon
PTFE
Magnified views showing structural differences of some membrane types
Page 59
In-Line Filters Provide Good Insurance Against System OverPressure
NEW RRLC In-line filter and fitting – max 600 bar
Protect RRHT columns with efficient in-line filter with 0.2 µm pore size frits
Description Part number PorosityFrit diameter
Flow rate
Part number Replacement Frits
RRLC 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 mm, max 600 bar
5067-1553 0.2 µm 4.6 mm 1 - 5 mL/min
5067-1562 (10/pk)
Page 60
Summary of Fundamental Aspects Concerns of Routine Use of 1.8µm Columns
• Base Your Column and Instrument Choice on Typical Sample Needs
• Optimize Chemistry for Best Separation
• 1200 SL Flexible Capability Allows Better Asset Management by Running Current, Legacy and Extreme Methods
• Engineered Column Bed Design Minimizes Pressure and Allows Better Utilization of All Assets
• Use Filtration to Extend Column Life Time and Increase Instrument Up Time
• Attend to Detection Data Rates
• Optimize Instrument Volume
• Make Sure Instrument “Parts” are Compatible with High Performance Needs
Page 61
New Convenience for Column Installation in RRLC Instruments
Easy, hand tighten column connection with Polyketone fittings up to 600 bar PN: 5042-8957 (10/pk)
Page 62
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