column technologies for small and large molecule lc/ms ... · charlie van wandelen, luisa pereira,...

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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tage

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1000µl/min 800µl/min600µl/min400µl/min 200µl/min100µl/min

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Blow-up of the 80% Step Region

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tage

[mV

]

1000µl/min 800µl/min600µl/min400µl/min 200µl/min100µl/min80% step with different flow rates

Dwell volume ~150 µL

10

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).

15

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

17

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

36.73424.9

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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


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42.19804.2

47.43748.5

37.46650.2

36.73424.9

58.97460.339.09

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76.96445.162.91

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445.266.12444.9

58.72445.043.54

772.4 73.28445.0

56.95445.1

70.16445.1

55.47445.0


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


62.09% Coverage

RT: 19.55 - 80.08 SM: 5B

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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

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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

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400

600

800

1

O

OCH3CH3 CH3

O

HCH3

H

H

OH O

CH3

31

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)

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uAU

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1

3

64

5

7

Channel A UV

32

0 5

mAU

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200

250

300

350 Competitor DPyridine Tf – 1.46

0 5

mAU

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300

350

Competitor APyridine Tf - 1.46

0 5

mAU

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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]

100 150 200 250 300 350 400 450 500m/z

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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

34

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

36

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

38

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

39

Stationary phase – retention on C18 vs PGC

C18 Hypercarb®

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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

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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]

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0

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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

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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

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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

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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


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


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

column technologies for small and large molecule lc/ms ... · charlie van wandelen, luisa pereira,...

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