Column Technologies for Small and Large Molecule LC/MS
Method Development
ASMS 2004Nashville
Charlie van Wandelen, Luisa Pereira, Eric D. Stover
2
Why is the Peak Not the Same
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The Chromatographer’s Peak
Your System’s Result
3
Don’t Believe Anybody
born in Norwich, Connecticut, was originally a rebel who became a general in the Continental Army. He and Ethan Allen captured Fort Ticonderoga, gaining needed supplies for the Americans in the American Revolutionary War. Later in 1775 Arnold also led an expeditionary force from Boston to Quebec and participated in an unsuccessful attack in the Battle of Quebec (1775) In 1777, without a command of his own, he still played a role in defeating the British at the Battle of Saratoga. He was a very good strategist who was well liked by his men and a friend of George Washington. Arnold was a principled man who felt that the Revolutionary War should be a fight purely between Britain and her colonies. When General Washington and the Continental Congress made an alliance with France against Britain, he disagreed strongly and began to pass information to British forces.
Benedict Arnold
4
Top Five Reasons Your Results Differ
• There is no Chromatographer here
• When does the gradient Begin?
• Equilibration………..Don’t Bet on it
• Connections, Connections, Connections!!! Dispersion
• You Dissolved it in What?...High organic + TFA?
• Temperature of Column, Eluent?, Sample?
5
When was the last time they saw this?
6
Why is the Peak Not the Same
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The Chromatographer’s Peak
Your System’s Result
7
Gradient Delay / Dwell / Dead Volume
• Typical gradient dwell, delay, dead? volume and accuracy chromatogram
50% Level 50% Level
100% Level
A B A – Column t0 Void volumeB – Gradient dwell (delay) volume
Apparent onset of gradientFrom Inj to Det
B
8
0% to 100% Step Gradients
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1000µl/min 800µl/min600µl/min400µl/min 200µl/min100µl/min
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1000µl/min 800µl/min600µl/min400µl/min 200µl/min100µl/min
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Blow-up of the 80% Step Region
23 24 25 26 27 28Time [min.]
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[mV
]
1000µl/min 800µl/min600µl/min400µl/min 200µl/min100µl/min80% step with different flow rates
Dwell volume ~150 µL
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Decreased Gradient Delay with High Pressure Mixing
• High Pressure Mixing– Tends to have smaller gradient
delay volumes.– Typically good at lower flow
rates
• Low Pressure Mixing– Versatility– Performance can vary with
manufacturer
11
Is the Column Equilibrated?
Column Volumes
Relative Equilibration
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Now? Now? Now?
5.0
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Is the Column Equilibrated?
• Yes, When
• When the Retention time is Stable
• When the Peak Shape is Acceptable
13
Why is Controlling Dispersion Important in Chromatography?
• Dispersion reduces efficiency and resolution
• Dispersion causes dilution and loss of sensitivity
• Minimizing dispersion is something that we can do to enhance system performance
• Most of the dispersion must take place in the column in order to separate anything with high resolution
14
Connective Tubing and Dispersion
F/DLd101.36 t4t
32 −×=σ
Dispersion in an open tube is dependent on the inner diameter of the connective tube (dt), the length of the connection (Lt), the flow rate (F) and the diffusion coefficient (D). For gradient methods, this effect is crucial post-column (the connection between the column and the API interface).
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Optimum Capillary LC Tubing
• Square cut and polished end• No dead volume
• Rough cut end• Potential dead volume
PEEKsil® for columns and componentsA microscope or good magnifying glass is an important tool for inspecting connectors in capillary LC.
16
Effect of Tube on 2mm ID Column98289
Ref: Technical bulletin No. 9, Rheodyne, Inc.
a.426024%loss
b.223060%
c.80086%
0.010 inch ID
d.5580
e. 54802%
f. 458015%
0.005 inch ID
5cm long 20cm long 80cm long
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Position of Connector Volume is Important
98290
Ref: Technical bulletin No. 9, Rheodyne, Inc.
a b
dc
tube at the detector end tube at injector end
tube at detector end tube at injector end
gradient
isocratic
18
Dispersion Table
Column
ID mm Vm(uL) Flow Max L Max Vol Max L Max Vol Max L Max Vol
mL/min (cm) (uL) (cm) (uL) (cm) (uL)
4.60 1080 1.0000 35.314 17.894 565.020 71.575 4359.726 198.820
4.00 817 0.8000 28.051 14.214 403.815 51.154 3115.859 142.095
3.00 459 0.5000 12.777 6.474 204.432 25.897 1577.404 71.935
2.00 204 0.2000 6.310 3.197 100.954 12.789 778.965 35.524
1.00 51.1 0.0500 1.577 0.799 25.238 3.197 194.741 8.881
0.50 12.8 0.0120 0.411 0.208 6.573 0.833 50.714 2.313
0.30 4.59 0.0040 0.160 0.081 2.555 0.324 19.718 0.899
0.18 1.65 0.0014 0.057 0.029 0.920 0.117 7.098 0.324
0.10 0.511 0.0004 0.020 0.010 0.315 0.040 2.434 0.111
0.003” ID tube0.01” ID Tube 0.005” ID Tube
Calculations based on Lcol=100mm Ncol=10,000Porosity=0.65 k'=1Instrumental variance <<Column variance
Maximum allowable tubing length with various ID columns
Blue PEEK Red PEEK Natural PEEK
02399
19
Tubing Internal Diameters and Volumes02001
19.478 49.474 1575 1.575 0.062 8.107 20.593 1016 1.016 0.040 4.560 11.583 762 0.762 0.030 2.027 5.148 508 0.508 0.020 1.140 2.896 381 0.381 0.015
.507 1.287 254 0.254 0.010
.248 0.631 178 0.178 0.007
.182 0.463 152 0.152 0.006
.127 0.322 127 0.127 0.005
.081 0.206 102 0.102 0.004
.046 0.116 76 0.076 0.003
.020 0.051 51 0.051 0.002
.005 0.0130 25 0.025 0.001 µl/cmµl/inMicronsMillimetersInches
20
Effect of Excess Connective Tubing
Base peak chromatogram of 100 fmol Myoglobin tryptic digest chromatographed on a 100x0.100mm ID BetaBasic 18 capillary column. Total post column volume was 0.874 µL (excluding any possible connection unswept volume. Peptides identified are in red (39.02%)
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42.19804.2
47.43748.5
37.46650.2
36.73424.9
58.97460.339.09
471.4
77.47444.8
76.96445.162.91
997.549.83806.4 74.99
445.266.12444.9
58.72445.043.54
772.4 73.28445.0
56.95445.1
70.16445.1
55.47445.0
GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYNEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG
39.02% Coverage
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42.19804.2
47.43748.5
37.46650.2
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471.4
77.47444.8
76.96445.162.91
997.549.83806.4 74.99
445.266.12444.9
58.72445.043.54
772.4 73.28445.0
56.95445.1
70.16445.1
55.47445.0
GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYNEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG
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42.19804.2
47.43748.5
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76.96445.162.91
997.549.83806.4 74.99
445.266.12444.9
58.72445.043.54
772.4 73.28445.0
56.95445.1
70.16445.1
55.47445.0
GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYNEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG
39.02% Coverage
21
Minimizing Tubing Dispersion the Payoff
Base peak chromatogram of 50 fmol Myoglobin tryptic digest chromatographed on a 100x0.100mm ID BetaBasic 18 capillary column. Total post column volume was 0.0975 µL (excluding any possible connection unswept volume). Peptides identified are in red (62.09%)
RT: 19.55 - 80.08 SM: 5B
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44.42536.7
38.55425.1
40.33650.3
41.47471.5
GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYNEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG
62.09% Coverage
RT: 19.55 - 80.08 SM: 5B
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GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYNEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG
62.09% Coverage
22
Summary…Know your System Intimately
• Dead Volume or the Dwell Volume, Know It
• Temperature of Column, Eluent?, Sample?
• Equilibration………..Investigate It
• Connections, Connections!!! Don’t Trust Anyone
• Determine Your Efficiency
• Know the Sample
Column selection inLC/MS Method Development
ASMS 2004Nashville
24
Column selection
• Stationary phase
– Symmetrical peaks (accurate integration)
– Sharp peaks (good signal-to-noise)
– Resolution (specificity)
– Retention of polars (enable qualitative and quantitative analysis)
– Alternative selectivity
25
LC/MS Methods
• Low concentrations of volatile buffers– Highly pure silica– Reduced silanol activity– Rugged bonding chemistry
• Organic solvent required in mobile phase– Retentive phase– Retention of polar compounds
• Generic mobile phases– Unique selectivity
26
Peak Tailing….Peak Height
• Basic Pharmaceuticals– Secondary Interactions
– Loss in :– Peak Height– Resolution– Integration accuracy– Quantitative results
BasicAnalyte
Neutral Analyte
Tf = a + b USP(5%)2a
27
Peak Tailing….
min0 2 4
mAU
-10
0
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30
40
, g , , ( )
min0 2 4
mAU
-10
0
10
20
30
40
, g , , ( )
Tf5% Peak 1 = 2.06
Potential Variance Height Peak 2: 16.8%
Tf 5% Peak 1 = 1.02
Potential VarianceHeight Peak 2: <3.5 %
a) b)1. 1.
2. 2.
Rs = 2(t2 – t1) / (w1 +w2)
28
Diphenhydramine - Quintiles
Hypersil™ BDS
Hypersil™ GOLD
Method conditions:-
Mobile Phase : 10mM ammonium Formate (pH3.0) / Acetonitrile (55:45 %v/v)
Flowrate : 1.0ml/min (split to 300ul/min into detector)
Original Column : Hypersil BDS C18, 150 x 4.6mm x 5u
Injection volume : 80ul
Column Temp : 40oC
Detection : SIM MS 256.1 (Span 0.2amu)
Flow diversion (to waste) 0-2min.
Sample : -
Diphenhydramine, 6ng/ml in a physiological salt solution, injected neat. i.e. no dilution.
RSD on 6 replicate injections of a 6ng/ml standard :-
BDS C18 - 2.3%
Gold - 1.3%
29
Pyridine Peak ShapePyridine Peak Asymmetry Comparison
0.00 0.50 1.00 1.50 2.00 2.50 3.00
Sym m etry® C18
Luna® C18
Xterra® MS
Zorbax® XDB
HyPURITY™ C18
Hypers il™ GOLD
Peak Tf (5% USP)
Hypersil™ GOLD
HyPURITY™ C18
Competitor A
Competitor B
Competitor C
Competitor D
30
Simvastatin (Cholesterol Reduction)
Dimensions: Hypersil™ GOLD 100 x 0.32µm, 5µmMobile Phase: A: Water + 0.1% Formic acid
B: MeCN + 0.1% Formic acidGradient 5% - 100% B in 10mins Flow: 6µl/minTemperature: AmbientInjection volume: 60nlDetection: UV 248nmSample: Simvastatin
min0 5 10
mAU
0
200
400
600
800
1
O
OCH3CH3 CH3
O
HCH3
H
H
OH O
CH3
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Banned aromatic amines
Dimensions: Hypersil GOLDTM 150 x 2.1mm, 3µmPart Number: 25003-152130Mobile Phase: A: Ammonium acetate 25mM pH5
B: AcetonitrileGradient: 20 to 100% B in 10 minsFlow: 0.2 ml/minTemperature: 40ºCDetection: UV at 254nm
Analytes:1. 2,4-diaminotoluene2. 4,4´-oxydianiline3. o-toluidine4. 2-methoxy-5-methylaniline5. 2,4,5-trimethylaniline6. 4,4´-methylene-bis(2-chloroaniline)7. Unknown
0 2 4 6 8 10 12 14Time (min)
0
uAU
2
1
3
64
5
7
Channel A UV
32
0 5
mAU
0
50
100
150
200
250
300
350 Competitor DPyridine Tf – 1.46
0 5
mAU
0
50
100
150
200
250
300
350
Competitor APyridine Tf - 1.46
0 5
mAU
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50
100
150
200
250
300
350 Hypersil™ GOLDPyridine Tf – 1.00
Improved Sensitivity
33
Hypersil™ GOLD stability: +ESI
Blank Hypersil™ Gold
H2O+0.1%formic acid / MeCN +0.1%formic acid
NL:2.69E5{0,0} + c ESI sid=20.00 Full ms [ 80.00-750.00]
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182.2
141.2155.2
130.1100.1272.2231.2 343.4 391.4
NL:7.84E4{0,0} + c ESI sid=20.00 Full ms [ 80.00-750.00]
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141.2
155.2
182.2
231.2130.1
114.1272.2
255.2 391.4102.2
419.5
No Ligand Bleed
m/z 328
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Retention of polars in RP-LC
• Solutions:– Highly aqueous mobile phase and long column lengths (small effect
on capacity factor)– Ion pair ( column equilibration times maybe long; not compatible
with all detection techniques )– HILIC (mechanism not fully understood)– Stationary phase which provides specific interaction mechanism for
polar compounds
• Cause: limited interaction of polar compounds with hydrophobic stationary phases
• Effect: reduced retention, thus low capacity factors• Consequences: inaccurate qualitative or quantitative analysis
35
PGC Surface Comparison
Dissolution of silicaat pH > 9
Cleavage oforganosilane at low pH
No surface groupsavailable forchemical attack
Stable acrosspH range 1 - 14
Schematic of Hypercarb™ vs Alkyl Silica:
Scanning Electron Micrograph of the Graphite Surface
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PGC - Polar Solute Retention
(a) Analyte with electron-withdrawing properties approaching the graphitesurface
(b) Analyte with electron-donating properties approaching the graphitesurface
Charge induced dipole
at the graphite surface
Charge induced dipole at the graphite surface
01177
Schematic Representation of a Point Charge Approaching the Graphite Surface
37
PGC - Polar Retention Effect
Courtesy of: V.Cocquart & M-C Hennion, 1992
Hamilton PRP-1 Hypercarb™
01176
Log P: Phenol 1.46Resorcinol 0.8Phloroglucinol 0.16
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Polar Retention Comparison
OH
OHHO
Polar Retention
0.00 0.50 1.00 1.50 2.00 2.50
C18
Perfluorinated
Polar endcapped C18
Polar embedded
Hypercarb
K' Phloroglucinol
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Stationary phase – retention on C18 vs PGC
C18 Hypercarb®
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100
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1001,2,3,4
0.30
0.31
0.31
0.36
TIC MS
m/z= 241.8-242.8
m/z= 242.8-243.8
m/z= 281.8-282.8
m/z= 265.9-266.9
0.0 1.0 2.0 3.0Time (min)
50
100
50
100
50
100
Rela
tive
Abun
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e 50
100
50
1004
321
0.79
1.13
3.55
3.70
TIC MS
m/z= 241.8-242.8
m/z= 242.8-243.8
m/z= 281.8-282.8
m/z= 265.9-266.9
Analytes: 1. Cytidine; 2. Uridine; 3. Guanosine; 4. Adenosine
Nucleosides
40
Stationary phase - retention on C18 vs. PGC
min10 200
AQUASIL C18
1
2
3
4
K775-1012
min10 20
12
3 4
PGC
H350-1040
0
AQUASIL C18 5µm, 100x0.32mm KAPPA®
Gradient: A - 20mM NH4 acetate, pH 5.5B - ACN0-10%B from 3 to 13 min
Flow:4µL/minTemp: 25°CDetection:UV @ 254nm
Hypercarb 5µm, 100x0.32mm KAPPAGradient:A - 20mM NH4 acetate, pH 5.5
B - ACN10-30%B in 15min
Flow:6µL/minTemp: 25°CDetection:UV @ 254nm
Sample: 1. 3´,5´-cCMP2. 3´,5´-cUMP3. 3´,5´-cGMP4. 3´,5´-cAMP
Nucleoside 3´,5´ - cyclic monophosphates
AquaSil™ Siliconizing Fluid fro treating glass surfaces is old by Pierce Chemical Company Co., Rockland. IL.
41
Hypercarb™: no phase bleed
• Phase bleed problems:– Decreased sensitivity– Source / lenses contamimation– Ligand may be isobaric with analyte
01347
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+ c ESI sid=20.00 Full ms [ 60.00-750.00]
100 150 200 250 300 350 400 450 500m/z
0
20
40
60
80
100 155.4
141.4
182.4
114.4
100.4
391.4223.3
+ c ESI sid=20.00 Full ms [ 60.00-750.00]
100 150 200 250 300 350 400 450 500m/z
0
20
40
60
80
100
Rel
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143.4
155.4141.4
114.4
100.4182.3
223.2391.4
Blank Column
42
Fosfomycin (antibacterial)
Column: Hypercarb™ 3µm 100x2.1mm
Mobile phase: A - H2O+ 0.1%ammonia
B - ACN
Isocratic (A:B) 90:10
Flow rate: 0.15ml/min
Temperature: 30°C
Detection: Negative ESI-MS (450°C, 3.5kV, 18V)
Analyte: Fosfomycin0.0 1.0 2.0 3.0 4.0
Time (min)
0
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Rel
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1.62 TIC MS
O
CH3 P OHO
OH
Log Pestimated (Fosfomycin) : -1.23
43
Glucosamine sulphate (antiarthritic)
Column: Hypercarb™ 3µm 100x2.1mm
Mobile phase: A - H2O+ 0.1%ammoniaB - ACN
Isocratic (A:B) 50:50
Flow rate: 0.2ml/min
Temperature: 60°C
Detection: Negative ESI-MS (450°C, 3.5kV, 22V)
Analyte: Glucosamine sulphate
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MS
O
NH2
OH
HO3SO
OH
OH
44
Acrylamide: C18 Silica vs PGC
0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8Time (min)
Hypercarb
BDS C18K’=0.6
K’=3.6
Mobile phase : H2OFlow rate: 400 µl/minInjection vol.: 10µl of acrylamide solution (0.5ng/µl)Detection: + Electrospray ( 500°C, 4.5kV, 20V )Scan: SIM [M+H]+ (m/z 72)
Acrylamide Calibration CurveR2 = 0.9993
0.0E+005.0E+051.0E+061.5E+062.0E+062.5E+063.0E+063.5E+064.0E+06
0 200 400 600 800 1000 1200
Concentration (ng/ml)
Are
a
Columns : Hypersil™ BDS C18 5µm,50x2.1mm
Hypercarb™ 5µm, 50x2.1mm
Calibration curve on Hypercarb for standards from 1 to 1000 ng/ml:
Ref.: J. Rosén and K-E. Hellenäs, Analyst, 2002, 127, 880-882Log P (acrylamide) : -0.67
45
Oligosaccharides from a Glycoprotein ( A )
( B )
( C )
( D )
2 5
01 0 0
5 0
01 0 0
5 0
01 0 0
5 0
0
Rel
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unda
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0 1 0 2 0 3 0 4 0 5 0 6 0 7 0
a 1
a 2
a 3
a 4
a 5a 6
a 7
a 8
a 9
b 1b 2
b 3
b 4
b 5 b 6b 7
c 1c 2
c 3
c 4
c 5
c 6
d 1 d 2 d 3d 4
d 5
d 6d 7
d 8
d 9d 1 0 ,1 1 d 1 2
d 1 3d 1 4
d 1 5d 1 6
d 1 7d 1 8
d 1 9 d 2 0
b 8
( A )
( B )
( C )
( D )
2 5
01 0 0
5 0
01 0 0
5 0
01 0 0
5 0
0
Rel
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0 1 0 2 0 3 0 4 0 5 0 6 0 7 0
a 1
a 2
a 3
a 4
a 5a 6
a 7
a 8
a 9
b 1b 2
b 3
b 4
b 5 b 6b 7
c 1c 2
c 3
c 4
c 5
c 6
d 1 d 2 d 3d 4
d 5
d 6d 7
d 8
d 9d 1 0 ,1 1 d 1 2
d 1 3d 1 4
d 1 5d 1 6
d 1 7d 1 8
d 1 9 d 2 0
b 8
TIC of reduced N-linked oligosaccharides
Column: Hypercarb™ 5um, 100x1mmMobile phase:
A – Ammonium acetate 5mM, pH 9.6 + 2% MeCN
B - Ammonium acetate 5mM, pH 9.6 + 80% MeCNGradient: 5 to 40% B in 80 minFlow rate: 50µl/minDetection: +ESI
Sample from: A – RNase BB – desialylated rhEPOC – fetuinD - sialylated rhEPO
TICs courtesy of: Nana Kawasaki, National Institute of Health Science, Tokyo, Japan
46
Column selection summary
• Hypersil™ GoldSymmetrical peaks (accurate integration)
Sharp peaks (good signal-to-noise)
Resolution (specificity)
• Hypercarb™
Retention of polars (enable qualitative and quantitative analysis)
Alternative selectivity without complex mobile phases
Stable pH 0 – 14
Putting the H(igh) P(erformance) in HPLC/MS Methods
ASMS 2004Nashville
48
Putting the Pieces Together
• Solvent Delivery System (HPLC pump)– Gradient vs. Isocratic– Plumbing– System Suitability – delay volumes
• HPLC Columns– Select the best stationary phase available– Column lengths and inner diameters– Method Development Strategies
• …..with the ultimate goal of:– Method durability– Sensitivity– Reproducibility
49
Choosing the Proper HPLC Column ID
Column Diameter
4.6 mm
3.0 mm
2.1 mm
1.0 mm
300 µm
180 µm
75 µm
Flow Rate1.0
ml/min0.5
ml/min0.2
ml/min50
µl/min3.0
µl/min700
nl/min200
nl/min
Theoretical increase
1 2.3 5 21 235 653 3765
Column Diameter
4.6 mm
3.0 mm
2.1 mm
1.0 mm
300 µm
180 µm
75 µm
Flow Rate1.0
ml/min0.5
ml/min0.2
ml/min50
µl/min3.0
µl/min700
nl/min200
nl/min
Theoretical increase
1 2.3 5 21 235 653 3765
Column Diameter Column Diameter
4.6 mm4.6 mm
3.0 mm3.0 mm
2.1 mm2.1 mm
1.0 mm1.0 mm
300 µm300 µm
180 µm180 µm
75 µm75 µm
Flow RateFlow Rate1.0
ml/min1.0
ml/min0.5
ml/min0.5
ml/min0.2
ml/min0.2
ml/min50
µl/min50
µl/min3.0
µl/min3.0
µl/min700
nl/min700
nl/min200
nl/min200
nl/min
Theoretical increaseTheoretical increase
11 2.32.3 55 2121 235235 653653 37653765
DConcmax2Column 1
D 2Column 2
DConcmax2Column 1DConcmaxConcmax
2Column 1Column 1
D 2Column 2D 2Column 2Column 2
nanospraymicrospray
50
Factors to Consider When Choosing Column ID
• Sample Availability– Limited sample = small ID column– Small ID = smaller injection volumes vs. longer run times
• Efficiency of API ionization method– Flow rate dependence on ESI/NSI efficiency– Higher source temperatures for higher flow rates– Good compromise is a 2mm ID column (if sample available)
• Ability of pump to operate at flow rate optimal for column– Do you want to split your flow– Will post-column split flow work?– Excess system volume effect on gradient profile
51
Maximum Injection Volumes for HPLC Columns
• Data represents maximum injection volumes for isocratic methods
• Injection volumes depend on strength of injection solvent
Column ID Length Column Volume
Flow rate
Inj Vol
Traditional 4.6mm 25cm 4.1ml 1 ml/min 100 ul Minibore 2.0mm 25cm 783ul 0.2 ml/min 19 ul Microbore 1.0 25cm 196 ul 47 ul/min 4.7 ul Capillary 100-320 um 25cm 20 ul 4.9 ul/min 485nl Nanoscale <100um Up to 200cm 490 nL 120 nL/min 12 nl
• For gradient methods, injection solvent should be the same strength or weaker than mobile phase at start of gradient.
• Remember – sample focusing, column equilibration
52
API Methods – Choose Wisely
• Choose ESI (NSI) for large molecules (proteins/peptides), polar compounds, acids, bases
– Solution based chemistry important– Multiple charged species– Flow rates : low nL to ≅ 400µL/min
• Choose APCI for small molecules, thermally stable, non-polar molecules
– Gas phase ionization (think gas phase acidities)
– Accommodates higher flow rates and greater buffer concentrations.
53
Mobile phases – Column Selection
Sensitivity vs organic concentration in mobile phaseABA06_pH5_6_16 5/19/2004 11:50:44 AM
RT: 0.00 - 4.97 SM: 7B
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time (min)
20
30
40
50
60
70
80
90
100
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
SN: 18670BP: 263.30
SN: 31BP: 263.30
SN: 26BP: 263.30
SN: 16BP: 263.30
SN: 11BP: 263.30
SN: 367BP: 263.30
SN: 25BP: 263.30
SN: 16BP: 263.30
SN: 10BP: 263.30
SN: 10BP: 263.30
NL:5.81E6Base Peak F: MS ICIS ABA06_pH5_6_16
NL:5.99E4Base Peak F: MS ICIS aba06_ph5_6_10
80/20 ACN/H2O
60/40 ACN/H2OS/N = 367
S/N = 1867
54
How Do We Get There – “Practically”
• Most pumps form nice gradients down to about 50-100µL/min (which means a 1 or 2mm HPLC column).
• Split flow– Post Column: Can be used with parallel detection (UV and MS)
when sample abundance permits– Pre-column: Can be done manually or integrated into pumping
systems
• Post Column split:to to electrosprayelectrospraysourcesource
to waste, UV or to waste, UV or fraction collectorfraction collector
HPLC columnHPLC column
Zero dead volume TZero dead volume T--piecepiece
PEEK TubingPEEK Tubing
PEEK TubingPEEK TubingPEEK TubingPEEK Tubing
to to electrosprayelectrospraysourcesource
to waste, UV or to waste, UV or fraction collectorfraction collector
HPLC columnHPLC column
Zero dead volume TZero dead volume T--piecepiece
PEEK TubingPEEK Tubing
PEEK TubingPEEK TubingPEEK TubingPEEK Tubing
55
Pre-Column Split – Turn any HPLC into a Capillary LC System (50x50 rule)
From HPLC pump50 ul/min
To waste
800 nl/min to cap LC column
Low dead volrequirements begin here
Courtesy Andrew Guzzetta
56
The Heart of Flow Rate Reduction
Valco Tee RestrictionCapillary
50umX50cm
To Injector Valveand Column
127 um IDFrom HPLC Pump
Low flow deadvolume concerns start hereAny Length
This set-up works for columns ranging in diameter between 0.32 to 0.075 mm. Adjust ultimate flow rate at the pump. No need to change restriction capillary for columns that supply sufficient back pressure Columns are self regulating!!! Smaller ID columns supply more back pressure and thus deliver lower flow rates as would be the demand! It is not unusual for 0.2 and a 0.075 mm ID columns to run at the same pre-split flow rate. For columns that supply extremely low back pressure (0.200X30mm, short desalting columns) change the restrictor to a larger diameter tubing to supply a lower resistance .
Courtesy Andrew Guzzetta
57
Gradient Profiles at 1µL/min
• Same procedure to measure gradient delay for standard column ID’s. (250x0.180mm ID)
t0 ???
58
Homemade vs. Integrated Splitting
• Homemade flow splitter (slightly larger gradient delay)
• Integrated flow splitter (enough volume to cause gradient mixing
GVS (9,1) Ron,Capillary.michrom gradient,9,1Acquired Wednesday, April 21, 2004 11:59:41 AM
0
5
Res
pons
e
10 20 30 40Retention time
GVS (8,1) Ron,Capillary.michrom gradient,8,1Acquired Wednesday, April 21, 2004 11:00:13 AM
0
5
Res
pons
e
10 20 30 40Retention time
59
Of course….No Splitting is Optimal
2 minute gradient, 15µL/min, no splitting!
Column: 50x0.32mm ID HyPURITY C18 5µmGradient: 30% to 90% ACN in 2 minSample: 1 – Acetophenone
2 – Butyrophenone3 - Heptophenone
60
High Throughput “No Split” gradient
• 30 second gradient without splitting, 15µL/min
Column: 50x0.32mm ID HyPURITY C18 5µmGradient: 30% to 90% ACN in 30 secSample: 1 – Acetophenone
2 – Butyrophenone3 - Heptophenone 0.625µL peak volume
61
Using Capillaries Smaller than 320µm ID
• Operating a substantially lower flow rates– 180µm ID = 1 – 1.4µL/min– 100µm ID = 400-600nL/min– 75µm ID = 200-300nL/min
• Increase length of restrictor (or run pump at higher flow rate)– Consider solvent conservation
• Decrease nanospray emitter tip diameter– 180µm = 30µm tip (100 to 75µm ID)– 100 and 75µm = 10 or 5µm tip (25µ ID)– Smaller emitter tips plug easier
• Consider connective tubing between column and emitter– PICOTIP™ alternatives (New Objective Inc)
62
To Maintain 90% of Your Columns Efficiency
5.0000.0250.1020.050
11.20050.0002.8000.0560.2300.07520.00075.0002.1700.1000.4200.10045.00075.0004.8900.2250.9500.15064.80075.0007.0800.3241.4000.180
cmµmcmµLµL/minmm
Length 25
micron
ID Connective
TubingMax
LengthMax
VolumeFlow Rate
Column ID
63
Connections
• Adjustable, fingertight connectors are replacing older designs
• A poor connection causes poor chromatography
• As column ID becomes smaller, poor connections become more evident
99101
64
Steel Ferrule Variations 98283
Upchurch Catalog
65
Connectors Must Match End-fittings 98284
Ferrule CannotSeal
Tube Not SeatedCausing UnsweptVolume
Tube and FerruleSeated Correctly
66
Carryover
• Problematic when working with small quantities
• Carryover can lead to chromatographic interference and thus decrease method robustness
• Analyte specific– i.e. analyte characteristics can play a role in carryover
• Rinsing with high volumes of organic can decrease carryover levels
67
0
0.002
0.004
0.006
0.008
0.01
0 500 1000 1500 2000 2500
Rinse Volume (uL)
% C
arry
over
.
Standard Vespel
Vespel w / Teflon coating
PEEK w/o Teflon
Tefzel w/o Teflon
Standard Tefzel
Standard PEEK
Rinse-out Characteristics w & w/o Teflon CoatingReserpine, 75% MeOH/25% H2O
Note: Standard Rheodyne PEEK and Tefzel are factorysprayed with a Teflon coating while Vespel is not. Compliments of Rheodyne®
Valve Seal Compositions
68
0
0.05
0.1
0.15
0.2
0.25
0 100 200 300 400 500Marination time (seconds)
% C
arr
yove
r
3 min desorption
1 min desorption
Carryover Due to Slow Diffusion in a ValveNote: Marination time refers to the time the concentrated
sample is left in the valve before being injected.
Compliments of Rheodyne®
Diffusion in Valve
69
Rinse-out Characteristics with Different SolventsUsing Tetracycline as the Sample
Note: Spritz volume refers to the rinse volume used by an autosampler.
0.0000
0.0050
0.0100
0.0150
0.0200
0.0250
% C
arry
over
100%
MeO
H
100%
AcN
Below Limit of Detection 10
% A
cN/ 9
0% H
2O
75%
MeO
H/ 2
5 %
H2O
50%
AcN
/ 50
% H
2O
10 %
MeO
H/ 9
0 %
H2O
250 microliter spritz volume 1250 microliter spritz volume
Compliments of Rheodyne®
Autosampler Rinse Solvents
70
Rinse-out Characteristics of Aged ValvesReserpine, 75% MeOH/25% H2O; Rheodyne 7750 valve
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
0.01
0 500 1000 1500 2000 2500
Rinse volume (uL)
% C
arry
over
25K cycles valve #1
25K cycles valve #2
50 K valve #1
50 K valve #2
Compliments of Rheodyne®
How old is your valve?
71
Capillary Installation – NSI Source
72
Maximizing Instrument Performance
25 fmol Phosphorylase B100x0.075mm ID BioBasic 18
10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Time (min)
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e Abu
ndan
ce
98.38402.3
75.69671.7
92.95434.3
71.73670.247.98
836.4
70.65784.4
51.06722.2
79.50445.2
56.58442.454.17
721.146.29632.2
63.35500.4 76.81
671.7
44.75730.441.82
537.1 67.89416.238.39
559.736.15460.1
64.60798.6
32.17459.1
91.95478.4 96.82
446.330.78585.5
82.54502.2
46% Protein Coverage
73
The Take Home…………….
• Choose the best HPLC column for the job, remember:– Modern base materials (silica), bonding chemistries, etc.
– Choose the right pore size, column geometry.
• Method Development considerations– System suitability (valves, etc.)
– Mobile Phase and additives
• Minimize System Dispersion– Make good connections
– Minimize post column tubing volume