William Long, Maureen Joseph, James D. Martosella, and John W. Henderson Jr.Agilent Technologies, 2850 Centerville Rd., Wilmington DE 19808, USA
Pittcon 2008New Orleans
Abstract
Conclusion
Efficiency in Hi Res, Hi Speed LC
Phase OrthogonalityPhenyl Selectivity
Sub 2-micron particles deliver efficiency andproductivity
Rs = N
4
k'
k'+1(-1)
Plates Selectivity Retention
Column Length = N
Particle Size = N
LN
dp
To Maintain Rs:
e.g.: L/2 dp/2
To Maintain Rs:
e.g.: L/2 dp/2
This is the basic premise from which we operate.
Sub 2-Micron Columns Provide the Efficiency ofLonger Columns for More Productivity
2,500
6,500
12,000
17,000
24,000
32,500
ResolvingPower
N(1.8 µm)
N.A.
N.A.
4,200
6000
8,500
12,500
ResolvingPower
N(5 µm)
552,10015
1264,20030
2107,00050
32010,50075
42014,000100
55021,000150
TypicalPressure
Bar (1.8 µm)
ResolvingPower
N(3.5 µm)
ColumnLength(mm)
2,500
6,500
12,000
17,000
24,000
32,500
ResolvingPower
N(1.8 µm)
N.A.
N.A.
4,200
6000
8,500
12,500
ResolvingPower
N(5 µm)
552,10015
1264,20030
2107,00050
32010,50075
42014,000100
55021,000150
TypicalPressure
Bar (1.8 µm)
ResolvingPower
N(3.5 µm)
ColumnLength(mm)
AnalysisTime
PeakVolume
AnalysisTime
PeakVolume
-90%
-80%
-67%
-50%
-33%
AnalysisTime*
-90%
-80%
-67%
-50%
-33%
AnalysisTime*
SolventUsage
* Reduction in analysis time compared to 150 mm column• pressure determined with 60:40 MeOH/water, 1ml/min, 4.6mm ID
More RRHT Bonded Phase Choices Allow for OptimizedMethod Development
Conditions: Columns: RRHT 4.6 x 50mm, 1.8um Mobile Phase: Acetonitrile (40%):Buffer 60% pH=2.4 25mM NaH2PO4
Flow 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
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!
Selectivity Impacts Resolution Most
Different Selectivity of ZORBAX Phenyl ColumnsAmines from Azo Dyes
1. Analine2. o-toluidine3. Anisidine4. Chloroaniline5. Benzidine6. Naphthylamine7. o-tolidine8. Dimethoxybenzidine9. Dichlorobenzidine
909
250%Btime
909
250%Btime
Gradient: A: 10 mM NH4Acetate, pH 4.7, B: MeOH
min2 3 4 5 6 7 8 9
mAU
0
50
100 SB- Phenyl
1 2 3 45
6
7 8 9
min2 3 4
mAU
0
50
100XDB-Phenyl
8
12,3
45
6,7
9
min2 3 4 5 6 7 9
mAU
0
50
100
Eclipse Plus Phenhex 4.6 x 100 mm, 5 um
1 2 3 45
687 9
min2 3 4 5 6 7 8 9
mAU
0
50
100 SB- Phenyl
1 2 3 45
6
7 8 9
min2 3 4
mAU
0
50
100XDB-Phenyl
8
12,3
45
6,7
9
min2 3 4 5 6 7 9
mAU
0
50
100
Eclipse Plus Phenhex 4.6 x 100 mm, 5 um
1 2 3 45
687 9
min2 3 4 5 6 7 8 9
mAU
0
50
100 SB- Phenyl
1 2 3 45
6
7 8 9
min2 3 4
mAU
0
50
100XDB-Phenyl
8
12,3
45
6,7
9
min2 3 4
mAU
0
50
100XDB-Phenyl
8
12,3
45
6,7
9
min2 3 4 5 6 7 9
mAU
0
50
100
Eclipse Plus Phenhex 4.6 x 100 mm, 5 um
1 2 3 45
687 9
min2 3 4 5 6 7 9
mAU
0
50
100
Eclipse Plus Phenhex 4.6 x 100 mm, 5 um
1 2 3 45
687 9
Different Selectivity of ZORBAX Phenyl ColumnsNucleobases and their nucleosides
1 2 3 4 5 6 7 8
mAU
0
50
100
1 2 3 4 5 6 7
mAU
0
50
100
min1 2 3 4 5 6 7 8
mAU
0
50
100
Eclipse Plus Phenhex 4.6 x 100 mm, 5 um
XDB-Phenyl
SB- Phenyl
1
1
2
2
2
3
3
3
4
4
4
5
5
5
6
6
6
7
7
7
8
8
8
9
9
10
10
10
9
1
1 2 3 4 5 6 7 8
mAU
0
50
100
1 2 3 4 5 6 71 2 3 4 5 6 7
mAU
0
50
100
min1 2 3 4 5 6 7 8
mAU
0
50
100
Eclipse Plus Phenhex 4.6 x 100 mm, 5 um
XDB-Phenyl
SB- Phenyl
1
1
2
2
2
3
3
3
4
4
4
5
5
5
6
6
6
7
7
7
8
8
8
9
9
10
10
10
9
1
5061410
%Btime
5061410
%Btime
1. Cytosine2. Uracil3. Cytodine4. Guanine5. Uridine6. Adenine7. Thymine8. Guanosine9. Thymidine10. adenosine
A: 20 mM Ammonium Acetate pH 4.5, B: MeOH, F=1, λ=254 nm
Different Selectivity of ZORBAX Phenyl ColumnsEstrogens
1. Estriol2. estradiol3. ethynylestradiol4. Diethylstilbestrol (DES)5. dienestrol
min2 4 6 8 10 12
mAU
0
100
min2 4 6 8 10 12
mAU
0
100
XDB-Phenyl
SB- Phenyl1 2,3 4 5
min2 4 6 8 10 12
mAU
0
100
Eclipse Plus Phenhex 4.6 x 100 mm, 5 um
1 2 34 5
1 32
4,51. Estriol2. estradiol3. ethynylestradiol4. Diethylstilbestrol (DES)5. dienestrol
min2 4 6 8 10 12
mAU
0
100
min2 4 6 8 10 12
mAU
0
100
XDB-Phenyl
SB- Phenyl1 2,3 4 5
min2 4 6 8 10 12
mAU
0
100
Eclipse Plus Phenhex 4.6 x 100 mm, 5 um
1 2 34 5
1 32
4,5
min2 4 6 8 10 12
mAU
0
100
min2 4 6 8 10 12
mAU
0
100
min2 4 6 8 10 12
mAU
0
100
min2 4 6 8 10 12
mAU
0
100
XDB-Phenyl
SB- Phenyl1 2,3 4 5
min2 4 6 8 10 12
mAU
0
100
Eclipse Plus Phenhex 4.6 x 100 mm, 5 um
1 2 34 5
min2 4 6 8 10 12
mAU
0
100
Eclipse Plus Phenhex 4.6 x 100 mm, 5 um
1 2 34 5
1 32
4,5
Mobile phase: 60 % MeOH, F=1mL/min., λ= 220nm
Results of C18 vs. Phenyl with ACN and Methanol
R2SlopeSolventColumn BColumn A
0.98601.125AcetonitrilePhenyl HexylPhenyl Ethyl
0.98371.248MethanolPhenyl HexylPhenyl Ethyl
0.99120.9565AcetonitrilePhenyl PropylPhenyl Ethyl
0.97820.9062MethanolPhenyl PropylPhenyl Ethyl
0.97050.6768AcetonitrilePhenyl PropylC18
0.91760.5750MethanolPhenyl PropylC18
0.94220.7712AcetonitrilePhenyl HexylC18
0.92360.7718MethanolPhenyl HexylC18
0.96950.7011AcetonitrilePhenyl EthylC18
0.89080.6168MethanolPhenyl EthylC18
R2SlopeSolventColumn BColumn A
0.98601.125AcetonitrilePhenyl HexylPhenyl Ethyl
0.98371.248MethanolPhenyl HexylPhenyl Ethyl
0.99120.9565AcetonitrilePhenyl PropylPhenyl Ethyl
0.97820.9062MethanolPhenyl PropylPhenyl Ethyl
0.97050.6768AcetonitrilePhenyl PropylC18
0.91760.5750MethanolPhenyl PropylC18
0.94220.7712AcetonitrilePhenyl HexylC18
0.92360.7718MethanolPhenyl HexylC18
0.96950.7011AcetonitrilePhenyl EthylC18
0.89080.6168MethanolPhenyl EthylC18
Phenyl Ethyl = Eclipse Phenyl; Phenyl Propyl= StableBond PhenylPhenyl Hexyl = Eclipse Plus Phenyl Hexyl
Comparison of Eclipse Plus Family – C18, C8 Phenyl-HexylNSAIDS
Eclipse Plus Phenyl Hexyl
Eclipse Plus C8
Eclipse Plus C18
min0 2 4 6 8
mAU
0
100
200
300
400
min0 2 4 6 8 10
mAU
0
100
200
300
400
min0 2 4 6 8 10
mAU
0
100
200
300
400
1 2
3
4
5 6 7
1,2
3
4
5 6 7
1,2
3
4
5 6 7
Eclipse Plus Phenyl Hexyl
Eclipse Plus C8
Eclipse Plus C18
min0 2 4 6 8
mAU
0
100
200
300
400
min0 2 4 6 8 10
mAU
0
100
200
300
400
min0 2 4 6 8 10
mAU
0
100
200
300
400
1 2
3
4
5 6 7
1,2
3
4
5 6 7
1,2
3
4
5 6 7
Mobile Phase: 40 % ACN 60 % 25 mM Sodium Phosphate Buffer pH= 2.4 Flow Rate= 1.5 ml/min UV 210 nm 2µlElution order for Eclipse Plus Phenyl Hexyl: (1) Piroxicam, (2) Sulindac,(3) Tolmetin, (4) Naproxen, (5)Ibuprofen, (6) Diclofenac, (7) Celebrex (equal portions of approximately 1 mg/ml solutions
Each ZORBAX RRHT C18 Bonded Phases ProvidesDifferent Selectivity to Optimize a Separation
Eclipse Plus C18
EclipseXDB-C18
Extend-C18
StableBondSB-C18
Mobile phase: (69:31) ACN: waterFlow 1.5 mL/min.Temp: 30 °CDetector: Single Quad ESIpositive mode scanColumns: RRHT4.6 x 50 mm 1.8 um
Sample:1. anandamide (AEA)2. Palmitoylethanolamide (PEA)3. 2-arachinoylglycerol (2-AG)4. Oleoylethanolamide (OEA)
1 2
3
4
1 2 3 4 5
1 2
3
4
1 2 3 4 5
1 2
3
4
1 2 3 4 5
1 2
3
4
1 2 3 4 5
1 2,3 4
1 2 3 4 5
1 2,3 4
1 2 3 4 5
1 234
min1 2 3 4 5
1 234
min1 2 3 4 5
1st choiceBest Resolution& Peak Shape
2nd choiceGood alternate selectivitydue to non-endcapped
3rd choiceGood efficiency & peak shapeResolution could be achieved
4th choiceResolution not likely,Other choices better, forthis separation.
Multiple bondedphases for mosteffective results.
Separate More Peaks with 1.8 µmComplex Natural Product Extract from Ginseng
min13 14 15 16 17 18
mAU
50
100
150
200
250
300
350
min13 14 15 16 17 18
mAU
50
100
150
200
250
300
350 Agilent 1100 Series
400 bar pump
Agilent 1200 Series
600 bar pump
min13 14 15 16 17 18
50
100
150
200
250
mAU
min13 14 15 16 17 18
50
100
150
200
250
mAU
ZORBAX SB-C18, 2.1x150 mm 1.8 µm
ZORBAX SB-C18, 2.1 x 150mm, 5 µm
1.8 µm
5 µm
Improved Resolution
Agilent Application Note:Analysis of a complex natural productextract from ginseng – Part IPublication Number 5989-4506EN
Mobile phase: A = H2O + 0.1% TFA
B = ACN + 0.1% TFA
Gradient: 10% to 95% ACN in 40min,
hold for 1min
Flow: 0.4 mL/min
Inj. volume: 3 μ L, partial loop filling
DAD: 220 nm (20 Hz)
2µl flowcell
C18 Options for Orthogonality and Selectivity
CH
CH
CH
O
SiO
OH
O
O
Si
Si
CHCH
CH
CHCH
CH
Si
3
3
3
3
3
3
3
3
3
CH
CH
CH
O
SiO
OH
O
O
Si
Si
CHCH
CH
CHCH
CH
Si
3
3
3
3
3
3
3
3
3
O
R
R
Si
OH
Si
O
Si
R
R
R
O
OH
R
O
R
R
Si
OH
Si
O
Si
R
R
R
O
OH
R
CHCH
CH
CH
CHCH
O
SiO
O
O
O
Si
Si
Si
CHCH
CH
CHCH
CH
Si
3
3
3
3
3
3
3
3
3
3
3
3
Base silicaimproved Improved double endcapping
Eclipse Plus C18Eclipse XDB-C18
StableBond-C18
Recommended forhigh temperatures at low pH
R: diisobutyl
Extend C18
Designed for mid pHup to pH 11.5
OO
Si
Si
Extend C18
Designed for mid pHup to pH 11.5
OO
Si
Si
Scatter Plot Explanation
Hydrophobic interactions generally dominate reverse phaseHPLC; deviations from linearity can be attributed to othersolute interactions: π-π, dipole-dipole etc.
The scatter plot data shows Phenyl columns can takeadvantage of π-π interactions when using Methanol ratherthan Acetonitrile. The compounds used consist of nitrogensubstituted aromatics and polyaromatic hydrocarbons toclarify this point. Other compound groups were alsoexamined.
The suppressed retention of some aromatic compounds withAcetonitrile can be attributed to competitive π-π interactions.
Selectivity Differences used in Scatter PlotsNitro Substituted Aromatics
min0 1 2 3 4 5 6 7 8 9
mAU
0
20
40
min0 1 2 3 4 5 6 7 8 9
mAU
0
50
100
min0 1 2 3 4 5 6 7 8 9
mAU
0
20
40
min0 1 2 3 4 5 6 7 8 9
mAU
0
50
100
min0 1 2 3 4 5 6 7 8 9
mAU
0
10
20
30
min0 1 2 3 4 5 6 7 8 9
mAU
0
20
40
60
min0 1 2 3 4 5 6 7 8 9
mAU
0
10
20
30
min0 1 2 3 4 5 6 7 8 9
mAU
0
20
40
60
Eclipse Plus Phenyl Hexyl
Eclipse Plus Phenyl Hexyl
XDB-C18
XDB-C18
1. Nitrobenzene2. Dinitrobenzene3. Trinitrobenzene
60
%M
eth
an
ol
40
%A
ce
ton
itrile
12 3
123
1
2
3
Phenyl Phases can take advantage of π-π interactions andthus show different selectivity than C18s, which primarilyutilize hydrophobic interactions
y = 0.6803x + 0.0891
R2= 0.9686R2= 0.9686
40 % Acetonitrile 60 % Water
Nitroaromatics and PAHs Subset
-0.5
0
0.5
1
1.5
2
2.5
-1 -0.5 0 0.5 1 1.5 2 2.5 3
log k' Eclipse XDB-C18
log
k'Ec
lipse
Plu
sP
hen
ylH
exyl
y = 0.5839x + 0.1604
R2= 0.8857
60 % Methanol 40 % Water
Nitroaromatics and PAHs Subset
-0.5
0
0.5
1
1.5
2
-1 -0.5 0 0.5 1 1.5 2 2.5 3
log k' Eclipse XDB-C18
log
k'Ec
lipse
Plu
sP
hen
ylH
exyl
The new focus in LC columns is columns with sub 2-micron particles.These small particles provide very high resolution in short and traditionalcolumn lengths. But efficiency, N, is just one parameter in overallresolution in a separation. Selectivity has always been viewed as the mostimportant parameter in a separation. Different bonded phases anddifferent mobile phases provide changes in selectivity in a separation tooptimize resolution. Most chromatographers like to have the option of atleast 3 bonded phase choices available to obtain the needed resolution fortheir sample. These typically include C18, Phenyl and an alternate phasewith a different polarity. We can look at the need for these differentselectivity choices to manipulate resolution when columns with differentparticle sizes are used by comparing results with different samples withdifferent polarities and with varying numbers of analytes and calculatingpotential resolution.
RRHT columns make separations fast and provide superiorefficiency
Selectivity choices are critical for best results and isindependent of particle size.
Phenyl vs. other C18 phases in terms of orthogonality – Yes,it is different and worth trying for method development.
Phenyls vs. each other – range of choices means there isalways one to optimize your separation
Non endcapped phases such as StableBond C18 or Phenylare good alternate third phases for selectivity differences
Retention Comparison of Aliphatic and AromaticCompounds – C18 vs. Phenyl Phases
Experimental Process
•A series of over 40 aliphatic, nitro substituted benzenes and substitutedbenzenes were injected onto 4.6 x100 mm 5 micron columns using anAgilent 1200 system. The solvent consisted of either 40 % Acetonitrile 60 %Water or 60 % Methanol 40 % Water, at 1 ml/min, 205 nm. log k’ of eachcompound was determined and plotted.
•The scatter plot indicates that nitro substituted aromatics had thegreatest difference in retention on the C18 vs. Phenyl, but the aliphaticsand substituted benzenes also varied. This is shown by the correlationcoefficient of the plot.
•Therefore Phenyl columns are a good alternate selectivity to chooseduring method development.
•A variety of phenyl columns were also compared and differences in all thecolumns are there, but the C18 vs. Phenyl shows the most difference. Amethanol organic modifier changes selectivity more than acetonitrile andcan be used to also change selectivity.
Typical Method Development Parameters:Effects of Selectivity, Efficiency and 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
Re
so
luti
on
Increase N
Increase Alpha
Increase k'
Selectivity Impacts Resolution Most
• Change bonded phase
• Change mobile phase
• Plates are easiest to increase
Rs = N½/4 (-1)/ k’/(k’+1)
Plates: 5000 10000 15000 20000 25000
Alpha: 1.10 1.35 1.60 1.85 2.1
k’: 2.0 4.5 7.0 9.5 12.0
Plates: 5000 10000 15000 20000 25000
Alpha: 1.10 1.35 1.60 1.85 2.1
k’: 2.0 4.5 7.0 9.5 12.0
Typical methoddevelopmentparameters
Phenyl Options for Orthogonality and Selectivity
CH
CH
CH
O
SiO
OH
O
O
Si
Si
CHCH
CH
CHCH
CH
Si
3
3
3
3
3
3
3
3
3
O
R
R
Si
OH
Si
O
Si
R
R
R
O
OH
R
CHCH
CH
CH
CHCH
O
SiO
O
O
O
Si
Si
Si
CHCH
CH
CHCH
CH
Si
3
3
3
3
3
3
3
3
3
3
3
3
ImprovedType Bsilica
Improveddouble endcapping
Eclipse Plus PhenylhexylEclipse XDB-Phenyl
StableBond-Phenyl
Recommended forhigher temperaturesand low pH
R: diisobutyl
ZORBAX Phenyl
CH
CH
CH
O
SiO
OH
O
O
Si
Si
CHCH
CH
CHCH
CH
Si
3
3
3
3
3
3
3
3
3
Type Asilica
Type Bsilica
Type Bsilica
Multiple Types of Phenyl Columns ProvideSelectivity Differences
A
B
B
B
SilicaType
ZORBAX Phenyl
SB-Phenyl
Eclipse XDB-Phenyl
Eclipse Plus Phenyl
Agilent Column
Non-endcapped increaseshydrophilic and silanolinteractions
NoPhenylpropyl
Original phenyl offering
Type A silica has differentselectivity.
YesPhenylethyl
Original Type B SilicaPhenyl Offering
YesPhenylethyl
Longer hexyl spacer addshydrophobicity to thebonded phase.
YesPhenylhexyl
What distinguishes it?EndcapPhenyl BondedPhase Type
A
B
B
B
SilicaType
ZORBAX Phenyl
SB-Phenyl
Eclipse XDB-Phenyl
Eclipse Plus Phenyl
Agilent Column
Non-endcapped increaseshydrophilic and silanolinteractions
NoPhenylpropyl
Original phenyl offering
Type A silica has differentselectivity.
YesPhenylethyl
Original Type B SilicaPhenyl Offering
YesPhenylethyl
Longer hexyl spacer addshydrophobicity to thebonded phase.
YesPhenylhexyl
What distinguishes it?EndcapPhenyl BondedPhase Type
