Tips and Tricks of Faster LC Analysis Without Capital InvestmentCapital InvestmentIt may be easier than you think!

A il t T h l i IAgilent Technologies, Inc.

July 14, 2009

Page 1

There Are Many Reasons to Reduce Analysis Time

• Run More Samples/Day

• Reduce Mobile Phase Use• Reduce Mobile Phase Use

• Reduce Mobile Phase Waste Disposal

• Better Utilize Existing Resources in Instruments and Personnel• Better Utilize Existing Resources in Instruments and Personnel

• Implement “HB5”* Work Schedule

(* “HB5” H B 5(* “HB5” Home By 5, Ray Lombardi, Agilent Technologies, Inc. 2007)

At what cost and difficulty?At what cost and difficulty?That depends on what you want to accomplish.

Page 2

How Fast Do You Want To Go?Your Answer Will Determine the Cost of Increased SpeedYour Answer Will Determine the Cost of Increased Speed

• 2 to 3 X increase in Speed – Easy

• 5-10 X Increase – May require instrument tweak

• 10 X + Increase – Will require instrument upgrade and tweak

What are the paths to LC method speed gains?

• Most speed from column and particle size optimization

• Instrument optimization will enable or boost those gains

The following discussion is designed to simplify the process; Often at no cost!

Page 3

How Do Small Particles Help Speed Up Methods?Help Speed Up Methods?

• Increase efficiency (N) in same length columny ( ) g

• Allow shorter column to match efficiency of longer ..column

• Provide expanded flow rate range without loss of ..efficiency

Page 4

Trick: Use Smaller Particles For More Efficient Separations Across a Wide Flow Rate RangeSmall Particle Columns including Monolith

0.0030Dimensions: 4.6 x 50/30/20mmEluent: 85:15 ACN:Water

/

ZORBAX 5.0μmZORBAX 3 5μm

0.0020

0.0025 Flow Rates: 0.05 – 5.0 mL/minTemp: 20°CSample: 1.0μL Octanophenone in Eluent

ate)

ZORBAX 3.5μmVendor A 2.5μmVendor B 2.0μmZORBAX 1.8μmMonolith

0.0015

TP (c

m/p

la Monolith

0 0005

0.0010HET

0.0000

0.0005

0 0 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0

Page 5

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0Volumetric Flow Rate (mL/min)

ISOCRATIC ISOCRATIC ELUTION

Page 6

ResolutionResolution

N k 1⎛⎜ ⎞⎟−⎛⎜ ⎞⎟

αR N kks =

4 11

+⎛⎝⎜

⎞⎠⎟⎛⎝⎜

⎞⎠⎟

ααk4 1+⎝ ⎠⎝ ⎠α

Tip: Short Column/Smaller Particle Maintains RsTrick: Tailor Column to Analysis Needs

ColumnLength

Resolving Power

N(5 )

Resolving Power

N(3 5 )

ResolvingPower

N(1 8 )

Typical Pressure

B (1 8 )

Analysis Time*

(mm) N(5 µm) N(3.5 µm) N(1.8 µm) Bar (1.8 µm)

150 12,500 21,000 32,500 >400

100 8 500 14 000 24 000 >400AnalysisTi 33%100 8,500 14,000 24,000 >400

75 6000 10,500 17,000 320

50 4 200 7 000 12 000 210

Time

PeakVolume

-33%

-50%

67%50 4,200 7,000 12,000 210

30 N.A. 4,200 6,500 126

15 N A 2 100 2 500 55

Volume -67%

-80%

-90%SolventUsage15 N.A. 2,100 2,500 55 -90%

* Reduction in analysis time compared to 150 mm column

Page 8

Note: pressure determined with 60:40 MeOH/water, 1ml/min, 4.6mm ID

Goal: Run Current Method 2X FasterTrick: ½ Column Length and Smaller Particle = ½ TimeTrick: ½ Column Length and Smaller Particle ½ Time

Rapid ResolutionStableBond SB-C18

StableBond SB-C184.6 x 150 mm, 5 μm1

1Mobile Phase: 45% 25 mM NaH2PO4, pH 3.055% MeOH

Fl R t 2 0 L/ i

StableBond SB C184.6 x 75 mm, 3.5 μm

4.6 x 150 mm, 5 μm

Flow Rate: 2.0 mL/min.Temperature: 35°CDetection: UV 254 nmSample: Cardiac Drugs

1. Diltiazem Analysis Time:5 2 i

532

53

2

2. Dipyridamole3. Nifedipine4. Lidoflazine5. Flunarizine

5.2 minAnalysis Time:11.7 min

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14Time (min)

4

0 1 2 3 4 5 6Time (min)

4

Page 9

Goal: Run Current Method 3X Faster Trick: 1/3 Column Length and Smaller Particle = 1/3 Time

Original methodZORBAX LC column Extend C18

mAU

Extend-C184.6 x 150 mm, 5 μm3 μL inj.

100

150

200

250

12 5

4

36

min0 2 4 6 8 10

0

50

2 5

mAU

80

100

120 ZORBAX RRHT column Extend C184.6 x 50 mm, 1.8 μm1 μL inj2 3

4

51 6

3x faster

-20

0

20

40

60

Mobile phase: (70:30) MeOH: 50 mM pyrrolidine buffer Flow = 1.0 mL/min, Temp. : ambient

1 μL inj.2 3 5 6

Page 10

min0 2 4 6 8 1020

How Long Can the Column Be with MeOH?System Pressure With MeOH/Water

P t f P (b )

4.6 x 50mm Column with 1.8um particles at 1 ml/min,30oC

Percentage of MeOH

Pressure (bar)

0 14210 169

Pressure curve

200

250

e 10 16920 20330 22940 2490

50

100

150

pressur

40 24950 25260 24070 216

0 20 40 60 80 100

percentage of MeOH

70 21680 18490 145100 98

•Use P of 4.6 x 50mm as baseline

•Multiply P by Lcol2 / Lcol1

•If P is lower than max P of Instrument

Page 11

100 98you can run that length

How Long Can the Column Be with ACN?System Pressure with ACN/Water

4.6 x 50mm Column with 1.8um particles at 1 ml/min,30oC

Percentage of ACN (%)

Pressure (bar)

0 14210 154

System pressure with the change ofpercentage of ACN

150

re

10 15420 15930 15640 150

0

50

100

0 50 100

Pressur

40 15050 13560 12070 104

Percentage of ACN

•Use P of 4.6 x 50mm as baseline70 10480 8890 70

•Multiply P by Lcol2 / Lcol1

•If P is lower than max P of instrument you can run that length

Page 12

100 59instrument you can run that length

More Resolution in Original Method than NeededTip: 1.8um, Very Short Column, Faster Flow 5-10X Faster p yIsocratic on instrument with 400bar pressure limit

4.6 x 250 mm, 5 μm29.65

12

3

4

Rs(1,2) = 4.8N 21848

min0 5 10 15 20 25 30

4.6 x 100 mm, 3.5 μm12 71

12

3

4

Rs(1,2) = 3.5

N=22680N=21848

.

4.6 x 30 mm, 1.8 μm min0 5 10 15 20 25 30

12.71

12

3

4

R (1 2) 3 3

N=11691

N=6568 6 30 , 8 μ1 mL/min

min0 5 10 15 20 25 30

4.15

1 2 3

4

Rs(1,2) = 3.3

N=6104

N=6568

4.6 x 30 mm, 1.8 μm2 mL/min

min0 5 10 15 20 25 30

0.5 1 1.5 2 2.52.09

2 3

4Rs(1,2) = 3.1

C l ZORBAX SB C18 M bil Ph 50% 20 M N H PO H 2 8 50% ACN Fl R t 1 L/ i T t RT

N=6460

N=6463

Page 13

Columns: ZORBAX SB-C18 Mobile Phase: 50% 20 mM NaH2PO4, pH 2.8: 50% ACN Flow Rate: 1 mL/min Temperature: RTDetection: UV 230 nm Sample: 1. Estradiol 2. Ethynylestradiol 3. Dienestrol 4. Norethindrone

Tip: Constant Linear Velocity Maintains EfficiencyTrick: Reduce Flow Rate When Reducing Column Diameter

1mAU

175

Mobile Phase: 25% methanol in 0.4% Formic Acid

75

100

125

150

ZORBAX SB-C18, 4.6 x 250 mm, 5 μm, 1 mL/min

Solvent Used: 34 mL2

3

4

min0 5 10 15 20 25 30 35

0

25

50

mAU

2 45 6

min5 10 15 20 25 30 35U

100

125

150

175

ZORBAX SB-C18, 3.0 x 100 mm, 3.5 μm, 0.425 mL/minSolvent Used: 5.7 mL, decrease of 83% (decrease in analysis time of 57%)

0

25

50

75

Page 14

min0 5 10 15 20 25 30 35

Reference for Common Column DiametersMaintain Equivalent Linear Velocity for Different Column IDsMaintain Equivalent Linear Velocity for Different Column IDs

Column Type Column ID Flow RateypAnalytical 4.6 mm 1.0 mL/min

Solvent Saver 3.0 mm 0.42 mL/min

N B 2 1 0 21 L/ iNarrowBore 2.1 mm 0.21 mL/min

MicroBore 1.0 mm 47 μL/min

Capillary 0.5 mm 12 μL/minp y μ

Capillary 0.3 mm 4.2 μL/min

Nano 0.1 mm 472 nL/min

Flow rate column 2 = (diameter column 2)2/(diameter column 1)2 x Flow rate column 1

Nano 0.075 mm 266 nL/min

Page 15

Maintain equivalent mobile phase linear velocity when changing column diameter.

Isocratic Tips and Tricks Summary• Increased isocratic speed does not necessarily require ..sub-2um particles

P ti l di t d l l th j f t i• Particle diameter and column length are major factors in ..resolving power

• Chose new shorter column to approximate original column• Chose new, shorter column to approximate original column ..efficiency; Choose length based on time savings goal

– Example 3X: 150mm, 5µm (~12,000N) ~ 50mm, 1.8µm (~12,000N)p , µ ( , ) , µ ( , )

• Usually only need to reduce column length and particle size– Not necessary to change flow, injection vol. or organic content of MP

• Altered column diameter requires change in flow and injection ..volume

Page 16

GRADIENT GRADIENT ELUTIONELUTION

Page 17

Reducing Particle SizeReduces peak width; Increases height; DOES NOT change the gradient

mAU

16001.8um vs. 5um+ 60% Resolution

1200

1400 + 160% Efficiency

800

1000 Pressure at start: 51bar, 5µmRs(4,5) = 3.14Peak Width 5σ (9) = 0.107

200

400

600

Pressure at start: 394bar, 1.8µmRs(4,5) = 5.07Peak Width 5σ (9) = 0 074

min2 4 6 80

200 Peak Width 5σ (9) 0.074

Page 18

Note: Chromatograms offset for better view

Resolution Equation for Gradient Elution Relationship of k* to p

Gradient Time, Flow and Column Dimensions

Zorbax V = π x (Col internal radius)2 x Length x 0 6

VR ≈ N k*α

Zorbax Vm = π x (Col internal radius)2 x Length x 0.6

R ≈4

kα

Δ%B = difference between initial and final % B

tg Fk* ∝

Δ%B = difference between initial and final % B ...... S = constant (≈ 4 for 100 - 500Da)

F = flow rate (mL/min)tg = gradient time (min)

S (Δ%B) Vmg g ( )

Vm = column void volume (mL)

Page 19

Expected: Use of a Longer Gradient Time Increases Gradient RetentionIncreases Gradient Retention

4

2

1,21

3

5C l ZORBAX SB C8

Gradient Time

10 min.

Gradient Time60 min.

34 5 7

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

Gradient: 20 – 60% BMobile Phase: A:H2O with 0.1%TFA, pH 2

B: AcetonitrileFlow Rate: 1 0 mL/min

8

6,7

8

6

Flow Rate: 1.0 mL/minTemperature:35°CSample: Herbicides

0 25 500 5 10 15Time (min)Time (min)

8

Page 20

Increased gradient retention improves resolution of several peak pairs – 1,2 and 4,5

Tip: A Shorter Column (smaller Vm) Also Increases Gradient Retention and RsGradient Retention and Rs

1,2

4 6 150 5

4

5

Column: ZORBAX SB-C8

4.6 x 150 mm, 5 μm 4.6 x 75 mm, 3.5 μm

2

34

5

7

8Gradient: 20 – 60% BMobile Phase: A:H2O with 0.1%TFA, pH 2

B: AcetonitrileFlow Rate: 1.0 mL/minTemperature:35°CS l H bi id

6

8

1

3

67

Sample: Herbicides

0 5 10 15Time (min)

0 5 10Time (min)

I d di t t ti I ll l ti

Page 21

Increased gradient retention; Increases overall resolution

Tip: Improve Resolution By Using Short Column Length (Vm ) for Biomolecules

300SB C8 4 6 150 5 300SB C8 4 6 50 3 5

Length (Vm ) for Biomolecules

300SB-C8, 4.6 x 150 mm, 5µm 300SB-C8, 4.6 x 50 mm, 3.5µm

24 5

6 45

6

1

2

3

5

7 8

9

101

23

5

7 8

9

10

M bil Ph A 95% W t 5 % ACN 0 1% TFA S l 1 Gl T 6 L E k

0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16

Mobile Phase: A: 95% Water : 5 % ACN, 0.1% TFAB: 5% Water : 95% ACN, 0.085% TFAGradient: 10-60% B in 30 min.

Flow Rate: 1.0 mL / min.Temperature: Ambient

Sample: 1. Gly-Tyr 6. Leu-Enk2. Val-Tyr-Val 7. Angiotensin II3. [Gln11] Amyloid-β- 8. Kinetensin

Protein Fragm 1-16 9. RNase4. (TYR8) Bradykinin 10. Insulin (Eq.)5 M t E k

Page 22

5. Met-Enk

Tip: Maintain k* To Keep Relative Peak Position in a Chromatogram Unchanged While Reducing Timea Chromatogram Unchanged While Reducing Time

V - Column length Decrease in tG or F

Any Decrease in Can be Offset by a Proportional

Vm - Column length

V - Column (id)

G

Decrease in tG or FVm Column (id) Decrease in tG or F

k* ∝tG • F

S • Δ%B • Vm

Page 23

Trick: Change Vm and tg by Same ProportionTwo Chromatograms Both Having the Same Gradient Steepness

Column: Rapid Resolution

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

o C o atog a s ot a g t e Sa e G ad e t Steep ess

4

5

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

Gradient Time: 30 min.

Flow Rate: 1.0 mL/min4

5

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

Gradient Time: 15 min.

Flow Rate: 1 0 mL/min

8

8

Flow Rate: 1.0 mL/min

1

2

3AnalysisTime: 24 min 6

7

1

2

3

6

AnalysisTime: 12 min

0 5 10 15 20 25

77

Page 24

Time (min)

0 5 10 15 20 25

Time (min)

0 5 10 15

001784S1.PPT

Very Fast LC on Conventional 1100 HPLC Th I t tG1379 DegasserG1311 Quaternary pump

The Instrument

G1313A ALS autosamplerG1316A column compartmentG1314A VWD (standard cell G1314-60086, 10mm, 14uL)

AcetophenoneThe Sample

pDiethyl phthalateBenzophenoneButyrophenoneValerophenoneHexanophenoneHeptanophenoneOctanophenone

Page 25

Octanophenone

Page 26

Tip: Shorten Column & Gradient Time by Same FactorFactor1/3 Column Length- 1/3 Gradient TimeRRHT Column – 4.6 x 50mm, 1.8µm, SB-C18

VWD1A Wavelength=246nm(D:\SAMPLETEST\RRHT1100\HDS20070809172525\070809SBC180009D)Flow Rate 1.0 ml/min

mAU

120

140

VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS 2007-08-09 17-25-25\070809SBC180009.D)

1.1

76

2.2

5 4

3.3

57 Injection Volume 5uLTemperature 30°CWavelength 246nmSample rate 13 74 Hz

60

80

100

2.00

4

2.4

64

2.8

11

3.8

71

4.3

43

Sample rate 13.74 Hz

Time (min) % Acetonitrile

0

20

40

0 503.33 904.5 904 53 50

min0 1 2 3 4 5 6

0 4.53 505 50

Initial Pressure: 132 barFinal Pressure: 74 bar

Page 27

Final Pressure: 74 bar

The Comparison of Chromatogram with the Same RRHT column in 1100 and 1200 SLwith the Same RRHT column in 1100 and 1200 SL

VWD1 A Wavelength=246 nm (D:\SAMPLE TEST\RRHT 1100\HDS 2007 08 09 17 25 25\070809SBC180008 D)

Overlay the two Chromatograms

Norm.

120

140

VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS 2007-08-09 17-25-25\070809SBC180008.D) 1

.179

2.2

57

3.3

61

*DAD1 A, Sig=246,4 Ref=360,100 (WORKSHOP-TAIWAN\070815SBC180003.D)

80

100

2.4

68

6

40

60

2.0

07

2.8

1

3.8

74

4.3

43

min1 1.5 2 2.5 3 3.5 4 4.5 5

0

20

Page 28

The red one is 1200, the blue one is 1100.

Page 29

Gradient Tips and Tricks Summaryt Ftg F

S (Δ%B) Vm

k* ∝

Gradient Retention Relationship Formula Is the Key to:

( ) m

• Improving Resolution– Longer gradient time (tg)– Smaller column volume (Vm), length or ID– Keeping all other parameters constant

•Increasing Speed Without Losing Resolution•Increasing Speed Without Losing Resolution– Shorter column with proportionally shorter gradient time– Shorter gradient time with proportionally faster flow rate

Page 30

Shorter gradient time with proportionally faster flow rate

Tip: Method Translator Makes Changes EasyTrick: Let Agilent Method Translator Do the Math

Injection Volume Conversion

Basic Mode for Easy Transfer of Conventional Method to RRLC

Detector Settings recommendation

Gradient and Isocratic Method Conversion (auto-detected))

Page 31

Detailed OutputDetailed Input

Agilent Method Translator – Advanced ModeMore Detaled Information, But Still Easy to Use

pp

O i i l M th d N M th d

Page 32

Original Method New Method

Analysis of impurities of an active pharmaceutical ingredient by Does it work? - Example

conventional HPLC (4.6mm ID x 250 mm, 5.0 µm):mAU

OH

HN

CH3CH3

30

40 OH

OCH3

Main Compound

CH3CH3

20OH

HN

3C3

OCH3Impurity A

NCH3CH3CHCH

N33

H

NCH3CH3

H

10

Impurity A

O CH3

Br

Bromanisole

OCH3

Impurity B

OCH3

Impurity C

OH

OH

Impurity D

i0 2 5 5 7 5 10 12 5 15 17 5 20

0

Page 33

min0 2.5 5 7.5 10 12.5 15 17.5 20

Converting to a 4.6 mm ID x 100 mm, 1.8µm column:

Page 34

Does it work? Yes!mAU

30

40 Conventional HPLC

0

10

20

min0 2.5 5 7.5 10 12.5 15 17.5 20

mAU14

Resolution optimized 1.8µm

6

8

10

12

-2

0

2

4

Added Benefit of Increased Sensitivity!

Page 35

min0 1 2 3 4 5 6 7 82

mAU Conventional HPLC mAU35

Does it work?

30

40

25

30

mAU

20

2015

20

10

15

0

10

0

5

10

0

5

4.6 mmID x 250 mm, 5.0µm Zorbax SB C18

min0 2.5 5 7.5 10 12.5 15 17.5 20

0

4.6 mmID x 100 mm, 1.8µm Zorbax SB C18

min0 1 2 3 4 5 6 7 8

0

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

0

4.6 mmID x 100 mm, 1.8µm Zorbax SB C18 Speed Optimized0.00 min 5% B

20.00 min 90% B23.00 min 90% B23.01 min 5% B30 00 i 5% B

0.00 min 5% B8.00 min 90% B9.20 min 90% B9.21 min 5% B

12 00 i 5% B

Simple Conversion0.00 min 5% B4.33 min 90% B4.98 min 90% B4.99 min 5% B6 5 i 5% B

Speed Optimized

Page 36

30.00 min 5% B12.00 min 5% B6.5 min 5% B

ZORBAX Selectivity Choices Options for Improved Speed and ResolutionOptions for Improved Speed and Resolution

Reversed-Phase Chromatography Specialty d t

Extend-C18Bidentate-C18

StableBondDiisopropyl

Diisobutyl bonding

Eclipse XDBDimethyl bonding

Eclipse PlusDimethyl bonding

products

Double endcapping

SB-C18Diisobutyl C18

Diisobutyl bonding

Rx-C18

D gy g

Double endcapping

Eclipse XDB-C18SB-CNDiisopropyl-CN

D gy g

Double endcapping

Eclipse Plus C18Diisobutyl-C18

Rx/SB-C8Diisopropyl-C8

SB-C3Triisopropyl

DimethyloctadecylsilaneEclipse XDB-C8

Bonus-RPE b dd d id

Diisopropyl CN

Eclipse Plus C8

E li Pl

Eclipse PAH

SB-PhenylDiisopropyl-

Phenyl

SB-AQDiisopropyl

Eclipse XDB-Phenyl

E li XDB CN

Embedded amideDiisopropyl-C14

Triple endcappedEclipse Plus-Phenyl-Hexyl

Page 37

Eclipse PAHEclipse XDB-CN

Tip: Bonded Phase Can Change SelectivityImprove Peak Shape and Shorten HPLC Assay TimeImprove Peak Shape and Shorten HPLC Assay Time

• Each ZORBAX phase available in matching 1.8µm,3 5µm and 5µm choices most in 7µm!3.5µm and 5µm choices, most in 7µm!

• Over 140 RRHT,1.8um 600 bar column choices available

• 14 Different Column Chemistries – 13 bonded phases pand silica for HILIC use

• 7 column lengths (250* 150 100 75 50 30 and 20• 7 column lengths (250 , 150, 100, 75, 50, 30 and 20 mm long) * 1.8µm as custom column

3 i l di (4 6 3 0 2 1 d P ID)

Page 38

• 3 internal diameters (4.6, 3.0, 2.1 mm and Prep ID)

Tip: C8 Often Yields Same Peak Order in < Time Than C18 Trick: Evaluate Different Phases with Same MP

1. Oxybenzone

RRHT Eclipse Plus C18, 4.6 x 50mm, 1.8 umy

2. Internal Std.3. Octylmethoxycinnamate

N: 8800Pw: 0 014 min

N: 11600Pw: 0 047 min

N: 11800Pw: 0 093 min

247 bar

Rapid Resolution Eclipse Plus C8, 4.6 x 50mm, 3.5 um

Pw: 0.014 min.TF: 1.20

Pw: 0.047 min.TF: 1.06

Pw: 0.093 min.TF: 1.02

88 barmin0 1 2 3 4)

Conditions:Mobile Phase.: Water: Acetonitrile (30:70)Flow Rate: 2.0 mL/min. Detection: UV 230nm Temperature: 30 °C

2.2 min.N: 6600Pw: 0.037 min.TF: 0.99

N: 6300Pw: 0.061 min.

N: 5000Pw: 0.019 min.TF: 1.09

Temperature: 30 CSample: Lip balm extract in ACN(melted at 100°C ACN , cooled and 0.45 um filtered)

TF: 0.98

min0 1 2 3 4Less retention can save significant time – the C8 is a good choice here.The RRHT column is delivering the efficiency and resolution expected but the C8 bonded phase may be

Page 39

Group/Presentation TitleAgilent Restricted

Month ##, 200X

The RRHT column is delivering the efficiency and resolution expected, but the C8 bonded phase may be the best choice.

Tip: Polar Phases Can Save Analysis TimeTrick: Evaluate Non-Polar and Polar Phases with Same MP

Eclipse Plus Phenyl Hexyl

min0 2 4 6 8 10

Elution order reversal between Phenyl-Hexyl and Alkyl chains

Eclipse Plus C8

min0 2 4 6 8 10

y y y

Eclipse Plus C18

Mobile Phase 40 % ACN 60 % 25 mM Sodium Phosphate Buffer pH 2.4 Flow Rate: 1.5 ml/min 4.6 x 50mm UV 210 nm 2µl Elution order for Eclipse Plus Phenyl Hexyl: (1) Piroxicam, (2) Sulindac,(3) Tolmetin, (4) Naproxen, (5) Ibuprofen, (6)

min0 2 4 6 8 10

Page 40

Group/Presentation TitleAgilent Restricted

Month ##, 200X

µ p y y ( ) , ( ) ,( ) , ( ) p , ( ) p , ( )Diclofenac, (7) Celebrex (equal portions of approximately 1 mg/ml solutions

Tip: Higher Temperature Can Improve Speed and ResolutionSpeed and Resolution

Higher Temperature should always be considered

as a parameter during speed optimizationas a parameter during speed optimization

• Provides more rapid mass transfer:Improves Efficiency enhances resolution– Improves Efficiency – enhances resolution

– Decreases analysis time – faster separations with no loss in resolution

• Decreases mobile phase viscosityDecreases mobile phase viscosity– Lowers backpressure – allows for higher flow rates, faster separations,

greater efficiency

• Can change selectivity – optimize resolution

• Not necessary to go to the limit; Midrange offers benefits

Page 41

Tip: Temperature Changes Can Alter SelectivityTrick: Optimize Col Temp For Best Speed/Rs

20°CSalicylic acid

min0 1 2 3 4

30°C

0 1 2

0 1 2 345

3 4

Salicylic acidCoelution

40°C

0 1 2

0 1 2 345

Salicylic acid

60°C

0 1 2 345

0 1 2 345

Salicylic acid Column: RRHT SB-C184.6 x 50mm, 1.8um

90°C

0 1 2 345

345

Salicylic acid Increased T Decreases Run Time, Changes Selectivity and Sharpens Peaks

Improving Resolution

Page 42

0 1 2 345

Tips on When You Should Move to the Higher Pressure and Performance 1200 SL (RRLC) SystemPressure and Performance 1200 SL (RRLC) System • Very fast methods with high velocity mobile phase that increase backpressure above 400barp

• Resolution of difficult samples require long columns with 1.8µm particles (>400bar backpressure)

• Labs required to run both traditional and new, higher performance LC methods

• Need faster Data Acquisition for narrow, high efficiency peaks generated in higher speed methods

Page 43

Tip: 1.8µm Particle Increases Resolution; How?Trick: Substitute 1.8µm Particles in Same Length Column

Conditions for bothmAU 5Agilent 1100 Zorbax 2.1x150mm Conditions for both experiments

Pumps• Solvent A: H2O + 0.1% TFA

Solvent B: ACN + 0 1% TFA

mAU

250

300

350

17.1

36

15.9

67

59

15.5

55

15.0

65Agilent 1100 Zorbax 2.1x150mm SB C-18, 5µm

Solvent B: ACN + 0.1% TFA• Gradient: 10% to 95% ACN

in 40min, hold for 1min• Flow Rate: 0.4ml/minAutosamplers

100

150

200

15.4

1115.1

5Autosamplers• Injection volume: 3µlThermostatted Column

Comp.T t 50°C

Agilent 1200 RRLC250 2

mAU

079 Zorbax 2.1x150mm

SB C 18 1 8

min13 14 15 16 17 18

50

• Temperature: 50°CDetectors• DAD 2µl cell and 20Hz,

220nm, 150

200

250

2415.1

64 16.7

4615.4

7 215

.79015

. 0 SB C-18, 1.8µm

Ref: Appl. Note 5989-4506 by Edgar Naegele

50

100 15.3

2114

.937

14.7

87

Page 44

min13 14 15 16 17 18

Tip: Small Particles Improve Detection of Low Level Impurities

mAU

2

2.5

DAD1 A, Sig=254,4 Ref =of f (051119A\SIG10003.D) Column:150x4.6 mm

5µm

Pressure: 93 bar

N: 8213

4.6 x 150, 5um93 barN = 7259R S 1 15 20000

25000

30000

pdN 1

∝

p

0 5

0

0.5

1

1.5 Height: 1.25 mAU

S/N: 42.3

Rs = 1.15

tr = 14.9 min (1st epimer)

Nptp: 2.4 10-2 mAU

R_S = 1.15S/N = 42 Height = 1.25Noise = 24uAU

N

5000

10000

15000

20000 pd

min0 5 10 15 20

-1

-0.5

Norm.

DAD1 A, Sig=254,4 Ref=off (051007D\LC_X000001.D)

Norm.

DAD1 A, Sig=254,4 Ref=off (051007D\LC_X000001.D)Norm.

DAD1 A, Sig=254,4 Ref=off (051004D\LC_J0002.D)

Norm.

DAD1 A, Sig=254,4 Ref=off (051004D\LC_J0002.D)

Norm.

DAD1 A, Sig=254,4 Ref=off (051007D\LC_X000001.D)

Norm.

DAD1 A, Sig=254,4 Ref=off (051007D\LC_X000001.D)Norm.

DAD1 A, Sig=254,4 Ref=off (051004D\LC_J0002.D)

Norm.

DAD1 A, Sig=254,4 Ref=off (051004D\LC_J0002.D)1/dp

0.2 0.3 0.4 0.5 0.65000

Column:150 x 4.6 mm 1.8µm

Pressure: 490 bar

N: 28669

Height:1.78

S/N: 43.61.5

2

2.5

3Column:150 x 4.6 mm 1.8µm

Pressure: 490 bar

N: 28669

Height:1.78

S/N: 43.61.5

2

2.5

3Column:150x4.6 mm

3.5µm

Pressure: 165 bar

N: 14862

Height:1 34 mAU1

1.5

2

2.5 Column:150x4.6 mm

3.5µm

Pressure: 165 bar

N: 14862

Height:1 34 mAU1

1.5

2

2.5 Column:150 x 4.6 mm 1.8µm

Pressure: 490 bar

N: 28669

Height:1.78

S/N: 43.61.5

2

2.5

3Column:150 x 4.6 mm 1.8µm

Pressure: 490 bar

N: 28669

Height:1.78

S/N: 43.61.5

2

2.5

3Column:150x4.6 mm

3.5µm

Pressure: 165 bar

N: 14862

Height:1 34 mAU1

1.5

2

2.5 Column:150x4.6 mm

3.5µm

Pressure: 165 bar

N: 14862

Height:1 34 mAU1

1.5

2

2.5 4.6 x 150, 3.5um165 barN = 14862R_S = 1.37

4.6 x 150, 1.8um490 barN = 28669 R_S = 1.80 (+57%)S/N: 43.6

Rs = 1.80

tr = 17.2 (1st epimer)

Nptp: 3 10-2

-1

-0.5

0

0.5

1S/N: 43.6

Rs = 1.80

tr = 17.2 (1st epimer)

Nptp: 3 10-2

-1

-0.5

0

0.5

1Height:1,34 mAU

S/N: 50.7

Rs = 1.37

tr = 15.3 min (1st epimer)

Nptp: 2 10-2 mAU

-1

-0.5

0

0.5

Height:1,34 mAU

S/N: 50.7

Rs = 1.37

tr = 15.3 min (1st epimer)

Nptp: 2 10-2 mAU

-1

-0.5

0

0.5

S/N: 43.6

Rs = 1.80

tr = 17.2 (1st epimer)

Nptp: 3 10-2

-1

-0.5

0

0.5

1S/N: 43.6

Rs = 1.80

tr = 17.2 (1st epimer)

Nptp: 3 10-2

-1

-0.5

0

0.5

1Height:1,34 mAU

S/N: 50.7

Rs = 1.37

tr = 15.3 min (1st epimer)

Nptp: 2 10-2 mAU

-1

-0.5

0

0.5

Height:1,34 mAU

S/N: 50.7

Rs = 1.37

tr = 15.3 min (1st epimer)

Nptp: 2 10-2 mAU

-1

-0.5

0

0.5

_S/N = 50 Height = 1.34Noise = 20uAU

_ ( )S/N = 44 Height = 1.80Noise = 30uAU

Page 45

min101

min101

min0 5 10 15 201

min0 5 10 15 201

min101

min101

min0 5 10 15 201

min0 5 10 15 201

Particles Reveal More Information and Improve Detection and IntegrationDetection and Integration

7 Impurities7 Impurities4 Impurities

Customer Sample, Translation of Isocratic Impurity Methods, Zoom Critical Time Range (t = 7min)

7 Impurities

All 7 Baseline Separated!

7 Impurities

6 Not Baseline Separated!

4 Impurities

2 Not Baseline Separated!

4.6 x 150, 1.8μm490 barN = 28669

4.6 x 150, 3.5μm165 barN = 14862

4.6 x 150, 5μm93 barN = 7259

R_S = 1.80 (+57%)S/N = 44

R_S = 1.37S/N = 50

R_S = 1.15S/N = 42

Up to 60% higher resolutionwithout loss in sensitivity

Page 46

without loss in sensitivity

Example of What Is Possible!Shorter Column, Smaller Particle, > Flow Rate, High Temp

F= 1.20ml/minT = 40°CAnalysis Time = 11min

High Resolution:4.6mm x 150mm 5.0µm

, , , g pEasy!

Solvent Cons. = 13.2ml

min0 2 4 6 8 10 12

F = 4.80ml/minT = 40°CAnalysis Time = 1 05min

High Speed:Easy!

Analysis Time = 1.05minSolvent Cons. = 5.1ml

4.6mm x 50mm 5.0µm

min0 0.2 0.4 0.6 0.8 1

F= 1.00ml/minEasy!

T = 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

Max Speed at T = 95oC2.1mm x 50mm 1.8um

F= 2.40ml/minT = 95°CAnalysis Time: 0.4minSolvent Cons. = 1.0ml

PWHH = 197msec

> 20x faster !

Easy, But You Will Need > 400bar!

Page 47

min0.2 0.4 0.6 0.8 10

Tip: Higher Data Improves Response and Rs Trick: Optimize Data Rate for Accurate Profile

PW=0 30se 80Hz versus 10Hz Data Rate

Trick: Optimize Data Rate for Accurate Profile

80Hz

PW=0.30sec

80Hz versus 10Hz Data Rate• Peak Width: – 55%• Resolution: + 90%• Peak Capacity: + 120%• App Column Eff : + 260%

Data Rate

Peak Width

Resolution Peak Capacity

80 Hz 0.300 2.25 6040Hz

PW=0.33sec• App. Column Eff.: + 260%

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

20Hz

PW=0.42sec

PW=0 67sec 5 Hz 1.236 0.67 16

min0 1 0 2 0 3 0 4 0 50

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

Page 48

min0.1 0.2 0.3 0.4 0.50 Flow Rate: 5ml/min

Moving From Conventional LC to RRLC:“Doesn’t this

Moving to RRLC is simple and easy !Moving to RRLC is simple and easy !

• Same software (e g ChemStation EZChrom)

Doesn t this cost a fortune ?”

• Same software (e.g. ChemStation, EZChrom)

No additional training effort

• Limit capital investment by modular step-wise upgradep y p pg

Leverage existing 1100 modules, existing method

and stationary phase

• Configurable delay volume in 1200 SL pump

Easy to run legacy LC methods too

• Simple LC to RRLC method translator

No tedious method development“No - not at all !”

Page 49

Stepwise Scale-up to Rapid Resolution LCFrom 1100 to 1200 RRLC in two steps -Add Wh t Y N d Wh Y N d It!Add What You Need When You Need It!

Actual: 1100

Quat System

Step 1: 1100/1200

„Bin SL“ System

µ-Degasser

Step 2: 1200

Rapid Resolution System

Bin Pump SL

u-Degasser

Quat System

ALS

Quat Pump

Degasser

h-ALS SL

TCC

Bin Pump SL h-ALS SL

TCC SL

DAD SL

TCC

VWD/MWD/DAD

VWD/MWD/DAD+ Speed

+ Resolution+ Sensitivity

+ MS-Robustness+ Data Security & Traceability

+ Resolution+ Speed

+ MS-Compatibility

> 5 min> 6 min

> 1.5min> 2min

Analysis TimeCycle times

> 0.2min> 0.4min

y y+ Qualification (Degasser, ACE)+ Compatible with conv. HPLC

+ MS Compatibility+ Solvent Saving

+ Compatible with conv. HPLC

> 3 sec5 - 12,000

4.6 mm50 mm

0.2 - 10ml/min80 C

> 1.5 sec5 - 30,000

2.1 - 4.6 mm50 - 150 mm

0.05 - 5 ml/min80 C

Peak WidthN

Column IDColumn Length

Flow ratesT t

> 0.2 sec5 - 60,000

2.1 - 4.6 mm20 - 150mm

0.05 – 5 ml/min

Page 50

80 C400bar

80 C600 bar

TemperaturePressure

100 C600bar

SUMMARY

• Increasing speed of HPLC separations need not be difficult or costlycostly• Changes in columns and equipment depend on your goals• Majority of time saved will be based on column and particleMajority of time saved will be based on column and particle size• Isocratic and gradient 2X , 3X and 5X times savings are easy • Greater time savings may require instrument modifications• 10-20X increase in speed may require instrument upgrade• Long columns with 1.8µm particles may require >400 bar pump and autosampler capability

Page 51