A Guide To Speeding Up Your Separation - Agilent · A Guide To Speeding Up Your Separation. RRLC...

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A Guide To Speeding Up Your Separation

RRLC How-to GuideAgilent Restricted

June 11, 2008 of 26

Overview

• Choosing The Column

• Reducing The Column Length

• Changing The Column Id

• Adjusting The Injection Volume

• Transferring to 1.8um Columns

• Speeding It Up


June 11, 2008 of 26

Choose Suitable Column Phase

Mobile Phase pH

Stable Bond pH 1 – 4SB can use increased temperature up to 100°C

Acidic

Eclipse/Eclipse Plus pH 2 – 9

Eclipse can use increased temperature up to 60°C

Intermediate

Extend pH 8 – 11.5

Extend can use increased temperature up to 60°C

Basic


June 11, 2008 of 26

ZORBAX Portfolio of Modern Column Types for RP Chromatography – Eclipse Plus is Newest Family

SB-C18Diisobutyl-C18

Specialty products

Eclipse PAH

Extend-C18Bidentate-C18

Double endcapping

Extend-C18Bidentate-C18

Double endcapping

SB-PhenylDiisopropyl-

Phenyl

StableBondDiisopropyl

Diisobutyl bonding

Rx/SB-C8Diisopropyl-C8

SB-AQDiisopropyl

SB-C3Triisopropyl

Rx-C18Dimethyloctadecylsilane

Eclipse XDBDimethyl bonding

Double endcapping

Eclipse XDBDimethyl bonding

Double endcapping

Eclipse XDB-C18

Eclipse XDB-C8

Eclipse XDB-Phenyl

Eclipse XDB-CN

Bonus-RPEmbedded amideDiisopropyl-C14

Triple endcapped

Bonus-RPEmbedded amideDiisopropyl-C14

Triple endcapped

Reversed-Phase ChromatographyReversed-Phase Chromatography

SB-CNDiisopropyl-CN

Eclipse PlusDimethyl bonding

Double endcapping

Eclipse PlusDimethyl bonding

Double endcapping

Eclipse Plus C18

Eclipse Plus C8

Eclipse Plus Phenyl-Hexyl


June 11, 2008 of 26

Options for Quickly Changing Selectivity

CH

CH

CH

O

SiO

OH

O

O

Si

Si

CHCH

CHCH

CH

CH

Si

3

3

3

33

3

33

3

O

R

R

Si

OH

Si

O

Si

R

R

R

O

OH

R

CHCH

CH

CHCH

CH

O

SiO

O

O

O

Si

Si

Si

CHCH

CHCH

CH

CH

Si

3

33

3

33

33

3

33

3

Base silicaimproved Improved double endcapping

Eclipse Plus C18Eclipse XDB-C18

StableBond-C18

Recommended for high temperatures at low pH

R: diisobutyl

Extend C18

Designed for pH up to pH 11.5

OO

Si

Si


June 11, 2008 of 26

Different ZORBAX RRHT C18 Bonded Phases Maximum Selectivity

Eclipse Plus C18

EclipseXDB-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

Sample:1. anandamide (AEA)2. Palmitoylethanolamide (PEA)3. 2-arachinoylglycerol (2-AG)4. Oleoylethanolamide (OEA)

1 23

4

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.Match to one you are

currently using.


June 11, 2008 of 26

Reduce column length Shorter Columns with Smaller Particles

Rs = N4

k'k'+1

Plates Selectivity Retention

LN ∝

dp

To Maintain Rs:

e.g.: L/2 dp/2

••

000990P2.PPT

Column Length = N

Particle Size = N

Particle Size = P

α-1α


June 11, 2008 of 26

ColumnLength (mm)

ColumnEfficiency N(5 µm)

ColumnEfficiency N(3.5 µm)

ColumnEfficiency N(1.8 µm)

150 12,500 21,000 35,000

100 8,500 14,000 23,250

75 6000 10,500 17,500

50 4,200 7,000 12,000

30 N.A. 4,200 6,500

15 N.A. 2,100 2,500

Efficiency (N)

Pressure

AnalysisTime

PeakVolume

Analysis Time*

-

-33%

-50%

-67%

-80%

-90%

SolventUsage

•Reduction in analysis time compared to 150 mm column; all columns 4.6-mm i.d.• Shorter columns with small particles provide the efficiency of longer columns

with larger particles

Reduce column length


June 11, 2008 of 26

Comparison of Optimal Conditions On Columns with Different Dimensions – Length, Particle Size, Resolution

RRHT SB-CN 4.6 x 50 mm, 1.8um1.5 ml/min 1 µl injection

RRHT SB-CN 4.6 x100 mm, 1.8um1.5 ml/min 2 µl injection

1. Estril2. Estradiol3. Ethynyl Estradiol4. Dienestrol5. Diethylstilbestrol6. Ethynl estradiol methyl ester

SB-CN 4.6 x150 mm, 5um1.0 ml/min 3 µl injection

min2.5 5 7.5 10 12.5 15 17.5

mAU

0

25

50

75

100

125

150175

) 12 3

45

6

2.5 5 7.5 10

mAU

0

50

100

150

200

)

23

45

6

1 A: WaterB: 30 % MeOH/ 70 %MeCN40 % B Detection: UV 210 nm

2.5 5

mAU

-20

0

20

40

60

80

100

120140 2

34

56

1

RRHT Columns allow rapid resolution optimization during initial method development. Column lengths compared quickly.


June 11, 2008 of 26

Change Column I.D.?

Changing Column I.D.

Reduce Mobile Phase Use

Can improve speed potential – lower flow rate used – higher overheads

Can improve sensitivity for same mass loading


June 11, 2008 of 26

How conversion works for flow

Flow modification, for columns of different diameters

ml/minmmmmml/min 21.0

4.62.11.0 i.e.

2

=⎜⎜⎝

⎛⎟⎠⎞×

2 col.

2

column1

column21 col. Flow

.DiamDiam.Flow =

⎜⎜

⎝

⎛⎟⎟⎠

⎞×


June 11, 2008 of 26

Reduce column diameterand therefore flow rate

N.A. = not available

4.6 mm 3 mm

Reduce flow rate by factor of 0.4

Reducing to 3mm i.d. column allows use of higher linear flow rates as the 1200 SL pump will pump at 5ml/min.

2.1 mm4.6 mm

Reduce flow rate by factor of 0.2


June 11, 2008 of 26

Gradient Narrow-Bore HPLCReduces Solvent Waste and Improves Sensitivity

Mobile Phase: A: Water with 0.2% formic acid B: Methanol with 0.2% formic acidTemperature: 35°C; Inj: 2 μLSample: Organic Acids: 1.Gallic 2. Protocatechuic 3. Hydrocaffeic 4. Gentisic 5. Vanillic 6. Syringic 7. Sinapinic 8. Salicylic

Rapid Resolution SB-C184.6 x 30 mm, 3.5 μm

Gradient: 5-35% B in 6 min.Flow Rate: 1.0 mL/min

Solvent Used: 5.6 mL

Rapid Resolution SB-C182.1 x 30 mm, 3.5 μm

Gradient: 5-35% B in 6 min.Flow Rate:0.4 mL/min*

Solvent Used: 2.2 mL

*Flow rate is not proportional

• Instrument is optimized for lowest dwell volume and extra-column-volume

0

25

50

MAU

0.0 2.5 5.0Time (min)

1

2

3

4

56

7

8

0

50

100

150

MAU

Time (min)

1

2

3

4

5

6

7

8

0.0 2.5 5.0


June 11, 2008 of 26

Conversion for injection volume

Keep Injection volume proportional to column volume

2 col.column1

column21 col. Inj.Vol.

VolumeVolumeInj.Vol. =⎜

⎜⎝

⎛⎟⎟⎠

⎞×

2 col.column1

column21 col. 4

2.04.020 i.e. μlmlmlμl =⎜

⎜⎝

⎛⎟⎟⎠

⎞×

Zorbax column volume = 3.14 x r2 x L x 0.6 (r and L in cm)


June 11, 2008 of 26

Injection volume

RRHT Eclipse Plus Phenyl-Hexyl4.6 x 50 mm, 1.8um

RRHT Eclipse Plus Phenyl-Hexyl4.6 x 100 mm, 3.5um

Mobile Phase 40 % ACN 60 % 25 mM Sodium Phosphate Buffer pH= 2.4 Flow Rate= 1.5 ml/min UV 210 nm 2µl Elution 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

2µl injection

4µl injection

7.5

mAU

0

50

100

150

200

mAU

min2.5 5 7.5 10 12.5 15

0

50

100

150

200

Equivalent Resolution faster separation, shorter column


June 11, 2008 of 26

Update USP Assay for Fenprofen Calcium (non-steroidal anti-inflammatory (NSAID))

min0 1 2 3 4 5 6

mAU

025

5075

min0 1 2 3 4 5 6

mAU

025

5075

min0 1 2 3 4 5 6

mAU

025

5075

USP Requirements:L7 column, Rs > 8Tf(5%) < 2.0 for eachN=3000 or 20000/m4.6 x 150 mm, 5 um

5 μL inj.

Rapid Resolution4.6 x 100 mm, 3.5 um3.3 μL inj.

Rapid Resolution HT 4.6 x 50 mm, 1.8 um1.7 µL inj.

Mobile phase: ( 500:496:4) acetonitrile: water: H3PO4Flow = 2.0ml/min. isocratic Temp: ambient Detection: UV 272nm LC: Agilent 1100Sample: “resolution solution”, fenprofen (peak #1) with gemfibrozil prepared as described in USP

R = 16.5N/m=73000

R = 17.3N/m=117000

R = 15.8N/m=222000

Tailing factor for each of these six peaks is < 1.3

• High resolution and exceptional efficiency maintained for low cost updating to fast LC methods


June 11, 2008 of 26

Reduce injection volume

4.6 mm 3 mm

Reduction to allow for diameter change

2.1 mm4.6 mm

= 0.4 x Original

= 0.2 x Original

xReduction to allow for length change

150 mm 50 mm = 0.33 x Original

e.g. Original 4.6mm x 150mm transferred to 2.1mm x 100mm

= 0.2 x 0.67 = 0.13 x original injection volume

100 mm 50 mm = 0.5 x Original

150 mm = 0.67 x Original1100 mm


June 11, 2008 of 26

Injection Volumes for Different ID’s

Column ID Column Volume Peak Volume, k=1 Typical Injection Volume

Typical Inj Vol Range

4.6 mm 1500 μL 148 μL 20 μL 5 – 50 μL

3.0 mm 640 μL 44 μL 10 μL 3 – 30 μL

2.1 mm 320 μL 22 μL 2 μL 0.5 – 15 μL

1.0 mm 70 μL 4 μL 0.5 μL 0.1 – 3 μL

0.5 mm 15 μL 1 μL 150 nL 40 – 500 nL

0.3 mm 6 μL 0.3 μL 50 nL 15 – 250 nL

0.1 mm 700 nL 32 nL 10 nL 1 – 10 nL

0.075 mm 400 nL 18 nL 2 nL 0.5 – 5 nL

~Column Volume = 3.14 x (Column ID/2)2 x Column Length x 0.60Column length = 150 mm, N =13,000

• Typical injection volume = 10 – 30% of peak volume of first eluting peak.


June 11, 2008 of 26

How conversion works for time

Run Time or Gradient Segment Time Adjustment

.15250150.25 i.e. min

mmmmmin =⎜⎜

⎝

⎛⎟⎠⎞×

*assumes flow is proportional for columns 1 and 2

2 col.column1

column21 col. Time

LengthLengthTime =⎜

⎜⎝

⎛⎟⎟⎠

⎞×


June 11, 2008

0 10 20 30 40

Changing Gradient Time to AffectRetention (k*) and Resolution

Time (min)

100% B

100% B

100% B

100% B

tg= 40

tg= 20

tg= 10

tg= 5

000995P1.PPT

1/k* = gradient steepness = b

tg F

S D%B Vm

k* =

DF = 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

• S ≈ 4–5 for small molecules• 10 < S < 1000 for peptides

and proteins


June 11, 2008 of 26

Gradient Elution

This relationship also says:

If “b” is kept constant from run-to-runpeaks will elute in the same relative pattern.

1/k* ∝ = b = gradientsteepness

S • ΔΦ • Vm

tG • F


June 11, 2008 of 26

This Relationship Says that to Keep Relative Peak Position in the Chromatogram Unchanged

Column length

Column volume (i.d.)

ΔΦ (same column)

Decrease in tG or F

Increase in ΔΦ

Decrease in tG or F

Increase in ΔΦ

Decrease in tG or F

Any Decrease in Can be Offset by a Proportional

k* =tG • F

S • ΔΦ • Vm


June 11, 2008 of 26

Time (min)

0 5 10 15 20 25

1

2

4

5

3

Time (min)

0 5 10 15

Column: StableBond SB-C84.6 x 150 mm, 5 μm

GradientTime: 30 min.

Flow Rate: 1.0 mL/min

AnalysisTime: 24 min

8

6

7

1

2

45

3

8

6

7

Column: Rapid Resolution StableBond SB-C84.6 x 75 mm, 3.5 μm

GradientTime: 15 min.


AnalysisTime: 12 min

Sample: 1. Tebuthiuron 2. Prometon 3. Prometryne 4. Atrazine 5. Bentazon 6. Propazine 7. Propanil 8. Metolachlor

Two Chromatograms Both Having the Same Gradient Steepness


June 11, 2008 of 26

4.6 x 50 0.5 mL 5 min4.6 x 30 0.3 mL 3 min4.6 x 15 0.15 mL 1.5 min

4.6 x 150 1.54 mL 15 min

2.1 x 50 0.10 mL 5 min 60 sec2.1 x 30 0.06 mL 3 min 36 sec2.1 x 15 0.03 mL 1.5 min 18 sec

at 0.2 mL/min at 1.0 mL/min

Fast Re-equilibration with High Throughput RR and RRHT Columns

Column Volume (Vm) and Equilibration Time

Gradient Analysis Time = Run Time + Equilibration Timeusing single step return

Column Internal Equilibration TimeDimension (mm) Volume (Vm) at 1.0 mL/min (Vm x 10 x F)


June 11, 2008 of 26

Convert Run Time (Gradient Time) to shorter column

150 mm 50 mm

Reduction to allow for length change

= 1/3 x Reduction

Flow Rate = As converted

Injection Volume = As converted

Run time = Reduced by length change reduction factor

Gradient Times = Reduced by length change reduction factor

e.g. 15mins 5mins

35-60% over 15mins 35-60% over 5mins


June 11, 2008 of 26

Length = 50 mm, 3.5 μmtG = 6 min

Reducing Column Length Reduces Run Time

Gradient Time α Column LengthColumn: Eclipse XDB-C8, 4.6 mm i.d. Gradient: 45-90% B in tG minutes Mobile Phase: A: 25 mM Na2HPO4, pH 3 B: Methanol

Temperature:35°C Flow Rate: 1.0 mL/min Sample: Cardiac Drugs: 1. Diltiazam 2. Dipyridamole 3. Nifedipine 4. Lidoflazine 5. Flunarizine

Length = 75 mm, 3.5 μmtG = 9 min

Samplesper day

130

0.0 2.5 5.0 7.5Time (min)

0.0 2.5 5.0Time (min)

1

23

4

5

1

2 3

4

5

Samplesper day

96


June 11, 2008 of 26

1

2 3

4

5

1

23

4

5

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Time (min)

0.0 0.5 1.0 1.5 2.0Time (min)

F = 3.0 mL/mintG = 2 min

F = 2.0 mL/mintG = 3 min

Increasing Flow Rate Reduces Gradient Run Time Further

Column: Rapid ResolutionEclipse XDB-C84.6 x 50 mm, 3.5 μm

Gradient: 45-90% B in tG minutes

Mobile Phase: A: 25 mM Na2HPO4, pH 3 B: Methanol


Temperature: 35°C

Sample: Cardiac Drugs1. Diltiazam2. Dipyradamole3. Nifedipine4. Lidoflazine5. Flunarizine

If tG x F = constant then the elution pattern is unchanged

Samplesper day

275

Samplesper day

390


June 11, 2008 of 26

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

0.0040

0.0045

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

5.0 μm

3.5 μm

1.8 μm

1mL/min

2.0 mL/min

HETP

(cm

/pla

te)

Interstitial linear velocity (ue- cm/sec)

Speeding It UpIncrease Linear Velocity

(Flow Rate)

Increase Flow Rate – Step 1Increase the flow rate by 100%

• Reduce run time by 50%

• Reduce Gradient segments by 50%

Example• 1ml/min > 2ml/min

• 30 minutes > 15 minutes

• 35-65% over 30 mins > over 15 mins


June 11, 2008 of 26

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

0.0040

0.0045

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

5.0 μm

3.5 μm

1.8 μm

1mL/min

3.0 mL/min

HETP

(cm

/pla

te)


Increase Flow Rate – Step 2• Increase the flow rate by another

100%

• Reduce original run time to 1/3

• Reduce original Gradient time to 1/3

Example• 1ml/min > 3ml/min

• 30 minutes > 10 minutes

• 35-65% over 30 mins > over 10 mins

Is resolution OK? Is Pressure OK? Keep Going!


(Flow Rate)


June 11, 2008 of 26

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

0.0040

0.0045

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

5.0 μm

3.5 μm

1.8 μm

1mL/min

4.0 mL/min

HETP

(cm

/pla

te)


Increase Flow Rate – Step 3• Increase the flow rate by another

100%

• Reduce original run time to 1/4.

• Reduce original Gradient time to ¼

Example1ml/min > 4ml/min

• 30 minutes > 7.5 minutes

• 35-65% over 30 mins > over 7.5 mins


(Flow Rate)

Is resolution OK？Is Pressure OK? Keep Going!


June 11, 2008 of 26

• When flow limit of pump is reached

Consider using a smaller i.d. column

• When pressure is approaching maximum (e.g. 550 bar)

Remember to allow for increase in pressure at non-ideal mix

• When resolution is no longer satisfactory

When to Stop

Methanol/water 40C

0.000.200.400.600.801.001.201.40

0 20 40 60 80 100 120

%Methanol

Visc

osity

ACN/water 40C

0.00

0.20

0.40

0.60

0.80

0 20 40 60 80 100 120

%ACN

Visc

osity


June 11, 2008 of 26

ToolsRRLC Compendium CD – Pub. No. 5989-7261EN

• 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


June 11, 2008 of 26

Method Translator

ToolsRRLC Compendium CD – Pub. No. 5989-7261EN

Additional Info on CD• 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)


June 11, 2008 of 26

All scaling calculations to transfer methods to RRHT, 1.8um particles are done using the Agilent MethodTranslator

For free - P/N5981-7261EN


June 11, 2008 of 26

Features of the Agilent MethodTranslatorBasic mode with certain pre-set parameters:

Enter the parameters of your existing method and the parameters of the desired column you would like to convert to.

1.

2.

3.

4.

5.

6.


June 11, 2008 of 26

Features of the Agilent MethodTranslatorAdvanced mode – all calculation parameters in your hands:

More to enter but much more information returned.

3.

4.

1. 2.

[mL] [mL]


June 11, 2008 of 26

System Constraints: Peak Dispersion, Flow and Pressure Limitations

• Increased flow, along with decreased particle size, will significantly increase operating pressure - check with supplier for specifications, precautions

• Reducing the column volume increases the negative effect of system bandbroadening on the separation

• Ensure all sources of extra column broadening are minimised (e.g. tubing, flow cell volume)

• Reduced peak durations will need faster data acquisition to maintain at least 20 points across the peak

• Possibly at cost of extra noise on older systems

• Increased flow rate may affect degasser performance, pressure stability – check with supplier for specifications, precautions


June 11, 2008 of 26

Summary

• Shorter 1.8um columns allow faster analysis with same resolving power as longer 5um columns

• Change column diameter if necessary to allow for lower solvent consumption and higher accessible velocity

• Adjust the injection volume to maintain the same mass loading on the smaller column

• Adjust run time and gradient to allow for the shorter column

• Speed up the flow rate until max flow or max pressure or loss of resolution


June 11, 2008 of 26

Questions?


June 11, 2008 of 26

Upcoming LC eSeminars

Getting the Most from your LC –Optimizing for Speed

June 12, 2008 – 1:00pm EDT

Practical Considerations for Improving HPLC Selectivity and Resolution for Protein and Peptide Separations

June 26, 2008 – 1:00pm EDT

Discovering and Identifying More Protein Targets in Less Time

August 28, 2008 – 1:00pm EDT

A Guide To Speeding Up Your Separation - Agilent · A Guide To Speeding Up Your Separation. RRLC...

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