More Speed and Resolution for Current Methods

A Simple “How –to –Guide” for Easier Method Transfer in theEasier Method Transfer in the

Current Lab Environment

Thomas J. WaegheColumns and ConsumablesAugust 28, 2007

Page 1

Key Points for Successful Transfer

1. Set Your Goals--Speed, Resolution, Both?

2. Choosing The Columng

3. Reducing The Column Length

4. Changing The Column i.d.4. Changing The Column i.d.

5. Adjusting The Injection Volume

6 Transferring to 1 8um Columns - Is and Grad6. Transferring to 1.8um Columns Is and Grad

7. Increase Resolution

8 Speeding It Up8. Speeding It Up

Page 2

Rule of Thumb

Maybe not always 100% Technically Correct but a very good easy alternative

Page 3

Column Choice is Key to Overall Success

• Choose Bonded Phase as Close to Original Method as Possibleas Possible

• Try Alternate Bonded Phases to Solve Peak Placement ProblemsPlacement Problems

• Choose Column Chemistry for Optimum pH Stability and Column Life

Page 4

Bonded Phase Structures Affect Selectivityand pH Ruggedness

StableBond C18 Extend C18

C18C18

and pH Ruggedness

SiO

SiO

C18C18

Eclipse C18 StableBond CN

Page 5

Start Method Development with RRHT Columns: Different ZORBAX RRHT C18 Bonded Phases for Max Selectivity

Eclipse Plus C18

M bil h (69 31) ACN t

1 23

41st choiceBest Resolution& Peak Shape

StableBond SB C18

Mobile phase: (69:31) ACN: waterFlow 1.5 mL/min.Temp: 30 °CDetector: Single Quad ESI positive mode scan Columns: RRHT

1 2 3 4 5

1 234

2nd choiceGood alternate selectivity

Eclipse

SB-C18 Columns: RRHT 4.6 x 50 mm 1.8 um

Sample:1. anandamide (AEA)2 P l it l th l id (PEA)1 4

min1 2 3 4 5

due to non-endcapped

3rd choice Eclipse XDB-C18

2. Palmitoylethanolamide (PEA)3. 2-arachinoylglycerol (2-AG)4. Oleoylethanolamide (OEA)

1 23

4

1 2 3 4 5

3 choiceGood efficiency & peak shapeResolution could be achieved

4th choiceMultiple bonded

Extend-C181 2,3 4

4th choiceResolution not likely,Other choices better, for this separation.

phases for most effective method development.Match to one you are

Page 6

1 2 3 4 5

Match to one you are currently using.

How To “Match” a Column to a ZORBAX RRHT ColumnColumn

General Phase Type Starting ZORBAX Choice

Typical “endcapped” C18 or C8 bonded phases, newer columns Eclipse Plus C18 or C8

Endcapped C18 or C8 columnsEndcapped C18 or C8 columns, older generation Eclipse XDB-C18 or C8

Non-endcapped columns StableBond C18

Older types of columns StableBond C18, C8 etc.

Aqueous “type” columns SB-AQ

CN or Phenyl SB-CN, SB-Phenyl

Page 7

Choose Suitable Column Phase

Mobile Phase pH

Stable Bond pH 1 – 6

Acidic

Stable Bond pH 1 6SB can use increased temperature up to 100°C

IntermediateEclipse Plus /Eclipse XDB + pH 2 – 9

Eclipse can use increased temperature up to 60°C

B i

Extend pH 8 – 11.5

Extend can use increased temperature up to 45°C

Basic

Page 8

The Complete List of RRHT 600 bar Columns – Jan 1Eclipse

Dimensions Eclipse Plus C18 Eclipse Plus C8 XDB-C18 Eclipse XDB-C8 Extend-C184.6 x 150 959994-9024.6 x 100 959964-902 959964-906 928975-902 928975-906 728975-9024.6 x 50 959941-902 959941-906 927975-902 927975-906 727975-9024.6 x 30 959931-902 959931-906 924975-902 924975-906 724975-9024.6 x 20 926975-902 926975-906 726975-9023 0 x 150 959994 302

2 Recommended Starting Choices:

3.0 x 150 959994-3023.0 x 100 959964-302 959964-306 928975-302 928975-306 728975-3023.0 x 50 959941-302 959941-306 927975-302 927975-306 727975-3023.0 x 30 924975-302 924975-306 724975-3023.0 x 20 926975-302 926975-306 726975-3022.1 x 150 959794-9022.1 x 100 959764-902 959764-906 928700-902 928700-906 728700-902

• 959941-902• 959741-902They are Eclipse Plus C18 Columns.2.1 x 50 959741-902 959741-906 927700-902 927700-906 727700-902

2.1 x 30 959731-902 959731-906 924700-902 924700-906 724700-9022.1 x 20 926700-902 926700-906 726700-902Dimensions SB-C18 SB-C8 SB-Phenyl SB-CN SB-AQ Rx-Sil4.6 x 150 829975-902 829975-906 829975-912 829975-905 829975-9144.6 x 100 828975-902 828975-906 828975-912 828975-905 828975-914 828975-9014 6 x 50 827975 902 827975 906 827975 912 827975 905 827975 914 827975 901

C18 Columns.

4.6 x 50 827975-902 827975-906 827975-912 827975-905 827975-914 827975-9014.6 x 30 824975-902 824975-906 824975-912 824975-905 824975-9144.6 x 20 826975-902 826975-9063.0 x 150 829975-302 829975-306 829975-312 829975-3053.0 x 100 828975-302 828975-306 828975-312 828975-305 828975-314 828975-3013.0 x 50 827975-302 827975-306 827975-312 827975-305 827975-314 827975-3013.0 x 30 824975-302 824975-306 824975-3053.0 x 20 826975-302 826975-3062.1 x 150 820700-902 820700-906 820700-912 820700-9052.1 x 100 828700-902 828700-906 828700-912 828700-905 828700-914 828700-9012.1 x 50 827700-902 827700-906 827700-912 827700-905 827700-914 827700-9012.1 x 30 824700-902 824700-906 824700-912 824700-905 824700-9142.1 x 20 826700-902 826700-906

(Main product page)

Page 9

(Main product page)(P/N List)

Fi t L t’ W k E S d G i !First, Let’s Work on Easy Speed Gains!

Page 10

ISOCRATIC ISOCRATIC ELUTION

Page 11

Simplified Method Transfer for Increased Isocratic SpeedIncreased Isocratic Speed

1. Shorten the Column Length

2. Reduce Particle Size

3. Maintain Flow Rate

4. For More Speed….

Wait, Hold the Line - Easy Gains First!

V F i J F Slid A !Very Fast is Just a Few Slides Away!

Page 12

Reducing Column Length and Particle SizeMaintains Resolution While Reducing Assay Time

ColumnLength (mm)

ColumnEfficiency N(5 µm)

ColumnEfficiency N(3 5 µm)

ColumnEfficiency N(1 8 µm)

Analysis Time*

(mm) N(5 µm) N(3.5 µm) N(1.8 µm)150 12,500 21,000 35,000

100 8 500 14 000 23 250Efficiency (N)

AnalysisTime

-

-33%100 8,500 14,000 23,250

75 6000 10,500 17,500

50 4 200 7 000 12 000

( )

Pressure

Time

PeakVolume

-50%

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

30 N.A. 4,200 6,500

15 N A 2 100 2 500

67%

-80%

90%

SolventUsage

15 N.A. 2,100 2,500 -90%

• 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

Page 13

p p y gcolumns with larger particles

First, Reduce Column Length

5um 1.8um

Reduce column length by factor of 3

Quite often original method will have more resolution than is actuallyQuite often original method will have more resolution than is actually needed and a reduction by 5 may be possible

3 5 1 83.5 um 1.8um

Reduce column length by factor of 2

Quite often original method will have more resolution than is actually needed and a reduction by 3 is possible

Page 14

Reduced Column Length and Particle Size Reduce Analysis Time

Original methodZORBAX LC column Extend C18

mAU

Both Separations Performed on Same Agilent 1200 SL HPLC

Extend-C184.6 x 150 mm, 5 μm

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 μm

2 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

2 3 5 6

Page 15

min0 2 4 6 8 1020

Reached Flow Rate Limit? Change Column I.D.

Changing Column I.D. Will Allow

Lower Flow Rate to Match Linear velocity

Can improve speed potential – lower initial flow rate used allowsCan improve speed potential – lower initial flow rate used allows higher overheads within the flow rate range of the instrument

Reduce Mobile Phase Use

Can improve sensitivity for same mass loading, but must optimize the flow path volume to minimize dispersion.

Page 16

How conversion works for flow

Flow modification, for columns of different diameters

2⎛ ⎞2 col.

2column2

1 col. FlowDiamDiam.Flow =⎜

⎜⎛

⎟⎟⎠

⎞×

column1.Diam⎜⎝

⎟⎠

ml/minmmmmml/min 21.0

4.62.11.0 i.e.

2=

⎜⎜

⎝

⎛⎟⎠⎞×

Page 17

⎝

Reduce Column Diameterand Flow Rate by Same Factorand Flow Rate by Same Factor

4.6 mm 3 mm

Reduce flow rate by factor of 2.4

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

Compared to 4.6 mm id column at 1 mL/min, you can operate a 3 mm id column at 0 42 mL/min and save solvent or reduce flow rate to MScolumn at 0.42 mL/min and save solvent or reduce flow rate to MS detector, or you can keep the flow rate at 1 mL/min, and have 2.4-fold increase in linear velocity

2.1 mm4.6 mm

Reduce flow rate by factor of 4.8

Page 18

Quick Reference for Changing to Common Column DiametersCommon Column Diameters

Maintains Equivalent Linear Velocity for Different Column IDs:Column Type Column ID Flow Rateyp

Analytical 4.6 mm 1.0 mL/min

Solvent Saver 3.0 mm 0.42 mL/min

N B 2 1 0 21 L/ iNarrow Bore 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 19

• Maintain equivalent mobile phase linear velocity when scaling down in column diameter.

Changing Both Column Length and Diameter Change from a 4.6 x 250 mm (5 um) to a 3.0 x 100 mm (3.5 um) Column.

1mAU

175

Mobile Phase: 25% methanol in 0.4% Formic Acid

75

100

125

150 ZORBAX SB-C18, 4.6 x 250 mm, 5 mm, 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 mm, 0.425 mL/minSolvent Used: 5.7 mL, decrease of 83% (decrease in analysis time of 57%)

0

25

50

75

Page 20

min0 5 10 15 20 25 30 35

OK, Let’s Run…More Isocratic SpeedMore Isocratic Speed

• Increase flow rate• If flow rate limit of instrument* is reached, reduce column diameter and

reduce flow rate to maintain linear velocity• If pressure becomes problematic, increase mobile phase/column

temperaturep• Increase flow rate as necessary or desired to within 80-90% of flow or

pressure limit* Flow Rate Limit is 5mL/min for Agilent Binary Pump 10mL/min Agilent Quaternary Pump (P < 200 bar)

Page 21

Flow Rate Limit is 5mL/min for Agilent Binary Pump, 10mL/min Agilent Quaternary Pump (P < 200 bar)

Speeding It Up-How Fast can We Go?Increase Linear Velocity (Flow Rate)

0.0045

Increase Linear Velocity (Flow Rate)

0 0025

0.0030

0.0035

0.0040

5 0

Step 1 (2x Original Flow)Increase the flow rate by 100%

• Reduce run time by 50%

0.0010

0.0015

0.0020

0.0025 5.0 μm

3.5 μmcm

/pla

te)

• Reduce gradient time segments by 50%

-0.0005

0.0000

0.0005

0 0 0 2 0 4 0 6 0 8 1 0 1 2 1 4 1 6

μm 1.8

μm 1mL/minH

ETP

(c

Example0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

2.0 mL/min

Interstitial linear velocity (ue- cm/sec)

p• 1ml/min > 2ml/min

• 30 minutes > 15 minutes

• 35-65% over 30 mins > over 15 mins

Page 22

Speeding It Up – Faster!Increase Linear Velocity

0.0045 Step 2 (3 x original flow)

Increase Linear Velocity (Flow Rate)

0 0025

0.0030

0.0035

0.0040

5 0

p ( g )

• Increase the flow rate by another 100%

0.0010

0.0015

0.0020

0.0025 5.0 μm

3.5 μmcm

/pla

te) • Reduce original run time to 1/3

• Reduce original Gradient time to 1/3

-0.0005

0.0000

0.0005μm

1.8 μm

1mL/minHET

P (c

Example• 1ml/min > 3ml/min

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

3.0 mL/minInterstitial linear velocity (ue- cm/sec)

• 30 minutes > 10 minutes

• 35-65% over 30 mins > over 10 mins

Is resolution OK ? Pressure OK? Keep Going!

Page 23

Is resolution OK ? Pressure OK? Keep Going!

Speeding It Up-FASTER!!Increase Linear Velocity

0.0045Step 3 (4 x original flow)

Increase Linear Velocity (Flow Rate)

0 0025

0.0030

0.0035

0.0040

5 0

Step 3 (4 x original flow)

• Increase the flow rate by another 100%

0.0010

0.0015

0.0020

0.0025 5.0 μm

3.5 μmcm

/pla

te)

• Reduce original run time to 1/4.

• Reduce original Gradient time to ¼

-0.0005

0.0000

0.0005μm

1.8 μm

1mL/minHET

P (c

Example1 l/ i 4 l/ i0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

4.0 mL/min

Interstitial linear velocity (ue- cm/sec)

1ml/min > 4ml/min

• 30 minutes > 7.5 minutes

• 35-65% over 30 mins > over 7.5 mins

I l i OK? P OK? K G i !

Page 24

Is resolution OK? Pressure OK? Keep Going!

Agilent 1200 Rapid Resolution System – SpeedConventional LC → UFLC → RRLC

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

High Resolution:4.6mm x 150mm 5.0µm

Conventional LC UFLC RRLCRs = 4

Solvent Cons. = 13.2ml

min0 2 4 6 8 10 12

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

High Speed:

Rs will be ~60% lower

thoughRs = 2.3

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/minT = 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 !

Page 25

min0.2 0.4 0.6 0.8 10

8X Faster Analysis on RRHT Columns with Easy Method Transfer – High Pressure not RequiredMethod Transfer High Pressure not Required

Column: ZORBAX Eclipse XDB-C18Mobile Phase: 95% ACN: 5% WaterTemp: AmbientRRHT

Conventional

Temp: AmbientInjection volume: 1 uL

RRHT4.6 x 50 mm 1.8 μm

Flow Rate: 3 mL/minPressure = 229 bar

Conventional4.6 x 150 mm 5 μm

Flow Rate: 1 mL/minP = 37 bar

mAU

60

80

20

40 1.7 min13.5 min

min0 2 4 6 8 10 12 140

Sample: Vitamin E – α, β, γ-tocopherols in gel capE li XDB C18 i d fi t h i f th d

Page 26

Eclipse XDB-C18 is a good first choice for many methods.

Reduce Analysis Time by up to 95% using Rapid Resolution HT ColumnsRapid Resolution HT Columns

4.6 x 250 mm, 5 μm29.65

12

3

4

Rs(1,2) = 4.8N=22680N=21848

min0 5 10 15 20 25 30

4.6 x 100 mm, 3.5 μm12.71

12

3

4

Rs(1,2) = 3.5N=11691

.

4.6 x 30 mm, 1.8 μm 1 mL/min

min0 5 10 15 20 25 30

4 15

12

3

4

Rs(1,2) = 3.3

N=11691

N=65681 mL/min

4.6 x 30 mm, 1.8 μm

min0 5 10 15 20 25 30

4.15

1 2 3

4

s( , )

R (1 2) = 3 1

N=6104

N=6463 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

4Rs(1,2) = 3.1

Columns: ZORBAX SB-C18 Mobile Phase: 50% 20 mM NaH2PO4, pH 2.8: 50% ACN Flow Rate: 1 mL/min

N=6460

Page 27

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

When to Stop

• When flow limit of pump is reached

Consider using a smaller i.d. column

Wh i hi i ( 550 b )• When pressure is approaching maximum (e.g. 550 bar)

Remember to allow for increase in pressure at viscosity maximum for mobile phase mixture

Wh l ti i l ti f t• When resolution is no longer satisfactory

Methanol/water 40CACN/water 40C

0 600.801.001.201.40

cosi

ty0.40

0.60

0.80

cosi

ty

0.000.200.400.60

0 20 40 60 80 100 120

%M th l

Visc

0.00

0.20

0 20 40 60 80 100 120

%ACN

Vis

Page 28

%Methanol

Decreased Column Volume May Require Conversion for Injection Volume

Keep Injection volume proportional to column volume

2colcolumn2

1col Inj.Vol.VolumeInj.Vol. =⎜⎜⎛

⎟⎟⎞

× 2 col.column1

1 col. j.Vo .Volume

j.Vo . ⎜⎝

⎟⎟⎠

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

2 col.column1

column21 col. 4

2.04.020 i.e. μlmlmlμl =⎜

⎜⎝

⎛⎟⎟⎠

⎞×

Page 29

column1⎝ ⎠

Reduce injection volume

4.6 mm 3 mm

Reduction to allow for diameter change

= 0.4 x Original

2.1 mm4.6 mm = 0.2 x Original

xReduction to allow for length change

150 mm 50 mm = 0.33 x Original150 mm 50 mm 0.33 x Original

100 mm 50 mm = 0.5 x Original

150 mm 100 mm = 0.67 x Original

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

= 0.2 x 0.67

100 mm 50 mm 0.5 x Original

Page 30

= 0.13 x original injection volume

Reduce Injection Volume as Column Volume is Reduced

mAU

5075

USP Requirements:L7 column, Rs > 8Tf(5%) < 2.0 for each

4.6 x 150 mm, 5 um5 uL inj.

R = 16.5N/m=73.000

min0 1 2 3 4 5 6

025

50

mAU

f( )N=3000 or 20000/m

Rapid ResolutionmAU

025

5075

p4.6 x 100 mm, 3.5 um3.3 uL inj.R = 17.3

N/m=117,000

Tailing factor for each of these six peaks is <1.3

min0 1 2 3 4 5 6

0

mAU

75

Rapid Resolution HT 4.6 x 50 mm, 1.8

Mobile phase: ( 500:496:4) acetonitrile: water: H3PO4Flow = 2.0ml/min. isocratic Temp: ambient Detection: UV 272nm LC: Agilent 1100

R = 15.8N/m=222000

min0 1 2 3 4 5 6

025

504.6 x 50 mm, 1.8 um1.7 uL inj.

272nm LC: Agilent 1100Sample: “resolution solution”, fenoprofen (peak #1) with gemfibrozil prepared as described in USP

Page 31

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

GRADIENT GRADIENT ELUTIONELUTION

Page 32

Simplified Method Transfer for Increased Gradient SpeedIncreased Gradient Speed

1. Shorten the Column Length

2 Adj t G di t Ti b F t2. Adjust Gradient Time by same Factor

3. Maintain Flow Rate

4. For More Speed… Just Wait a Little Longer.

Remember, Easy Gains First!

“Faster” is Just a Few Slides Away!

Page 33

Faster is Just a Few Slides Away!

What is Gradient Elution?Is It More Difficult to Increase Gradient Speed?p

Increasing the solvent strength = Increasing the % organic in the mobile phase

Linear solvent strength gradient = % B per min is a constant

90 90%90

%ACNΔφ = 80%t G = 40 min.50%

70%

gradient time

gradient change

10 } } } } Δφt G = 2%/min.

0 10 20 30 40 min.

30%gradient slope

F 20% h i ACN t i 10 i

} } } }Δt 1 = Δt 2 = Δt 3 = Δt 4

Page 34

For every 20% change in ACN, t is 10 min.

Changing Gradient Time to AffectRetention (k*) and Resolution

100% B

100% B

tg= 5

tg Fk* ∝

0% B

tg= 101/k* = gradient steepness = b

S Δ%B Vm

k ∝

100% B

t = 20

0% B

100% B

tg= 20 ΔΦ = change in volume fraction of B solvent

S = constantF = flow rate (mL/min.)t di t ti ( i )

0% B

0 10 20 30 40

tg= 40tg = gradient time (min.)

Vm = column void volume (mL)

0% B

Group/Presentation TitleAgilent Restricted

Month ##, 200X

0 10 20 30 40Time (min)

000995P1.PPT

How conversion works for time

Run Time or Gradient segment Time Adjustment*

⎛ ⎞2 col.

column21 col. Time

LengthLengthTime =⎜

⎜⎝

⎛⎟⎟⎠

⎞×

1 0⎛ ⎞

column1Length⎜⎝

⎟⎠

.15250150.25 i.e. min

mmmmmin =⎜⎜

⎝

⎛⎟⎠⎞×

*

Page 36

*assumes diameter is equal for columns 1 and 2

Convert run time (and gradient) to shorter column with no flow increaseto shorter column with no flow increase

Flow Rate = As converted

Injection Volume = As convertedInjection Volume = As converted

Run time = Reduced by length change reduction factor

Gradient Times = Reduced by length change reduction factor

150 mm 75 mm

Reduction to allow for length change

= 1/2 x Reduction150 mm

e.g. 18mins 9mins

Page 37

45-90% over 18mins 45-90% over 9mins

Short Columns Reduce Total Gradient Analysis TimeTime

Gradient Separation of Cardiac Drugs

A 4 6 x 150 mm 5 mm B 4 6 x 75 mm 3 5 mm

1

5

5

A. 4.6 x 150 mm, 5 mmEclipse XDB-C8tG= 18 min

B. 4.6 x 75 mm, 3.5 mmEclipse XDB-C8tG= 9 min

1

14 4Run Time 13 min 8 minEquilibration 15 min 7 minTime

4.6 x 150 mm 4.6 x 75 mm

2

23

348 samples/day 96 samples/day

Total Analysis 28 min 15 minTime

Time (min) Time (min)0 2.5 5.0 7.5 1.00 12.5 15.0 0 2.5 5.0 7.5

Page 38

Time to Have Fun…More Gradient Speed

• Reduce Particle Size to Increase Efficiency and Peak Capacity • Decrease Gradient Time and Increase Flow Rate by Same Factor• If Flow Rate limit of instrument* is reached, reduce diameter of column

and reduce flow rate to maintain linear velocity• Repeat Flow Rate Increase to Flow or Pressure LimitRepeat Flow Rate Increase to Flow or Pressure Limit• If pressure becomes problematic, increase mobile phase/column

temperature*

Page 39

* Flow Rate Limit 5mL/min for Agilent Binary Pump, 10mL/min Agilent Quaternary Pump (P < 200 bar)

Faster Gradient Analysis of Cardiac DrugsWhat Changes Did We Make to Accomplish This?What Changes Did We Make to Accomplish This?

Optimized Column and Gradient

5

1

4

Column: Rapid Resolution Eclipse XDB-C8,

4.6 x 50 mm, 3.5 μmMobile Phase: A: 25 mM Na2HPO4, pH 3

Run Time 1.8 minEquilibration 1 min

2

p 3B: MeOH

Gradient: 42 – 90% B in 3 minFlow Rate: 3 mL/minTemperature:35°CSample: Cardiac Drugs

TimeTotal Analysis 2.8 minTime

3

1. Diltiazem2. Dipyridamole3. Nifedipine4. Lidoflazine5. Flunarizine

480 Samples/day

Page 40

Time (min)0.0 0.5 1.0 1.5 2.0 2.5 3.0

Column Efficiency under Gradient Conditions –Concept of Peak Capacity.Standard concept of column efficiency, N (plates), is only appropriate for isocratic conditions. A more useful concept for the case of gradient conditions is Peak Capacity - the number of peaks that can be separated (at a specified resolution) in a given amount of time. It is another measure of column efficiency.

4

2 peaks fit

5

5 μm

1 8 μm

2 peaks fit

Pc = (1+ tG/w)for gradient 1.8 μm

3 peaks fit

for gradient conditions

Rs = +50%

3 peaks fit50% more!!

Page 41

Gradient Resolution and Shorter Columns -Faster Analyses for Complex Samples – Higher Peak Capacity, Pc = (1+ tG/w):

2.1x150mm, 5μmP/N 883700-922 120 peaks

60 min

Detection: UV 214 nm Sample: HSA Tryptic Digestk* = 12.0

min15 20 25 30 35 40 45 50 55

70 min gradient0.2 mL/min

60 min.

min15 20 25 30 35 40 45 50 55

2.1x50mm, 1.8μmP/N 822700-90210 min gradient0.5mL/min

125 peaks10 min.!

k* = 12.9

min3 4 5 6 7 8 9 10

2.1x50mm, 1.8μmP/N 822700-902 156 peaks!k* = 38.6

min5 10 15 20 25

P/N 822700 90230 min gradient0.5mL/min

p25 min

Page 42

min5 10 15 20 25Conditions: Mobile Phase A: Water w/ 0.1% TFA, B: ACN w/0.1% TFA, Gradient 2%B to 50%B, Temperature: 50°C

Peptide Map of Tryptic Digest of BSA run on Agilent RRHT Zorbax SB-C18, 2.1x50mm, 1.8µAgilent RRHT Zorbax SB C18, 2.1x50mm, 1.8µ

Gradient time10min

Peak Capacity10min 347

15 min 398Longer Gradient Times Increase Peak

22.5 min 441

Capacity

Page 43

Peak Capacity as a function of Gradient Time (tg) and Column Length on 1.8 µm RRHT Columnsand Column Length on 1.8 µm RRHT Columns

Gradient Time 50 mm 100 mm 150 mm

10 347 --- ---10 347 --- ---

15 398 --- ---

20 47720 --- 477 ---

22.5 441 --- ---

30 535 54030 --- 535 540

45 --- 587 610

67 5 69467.5 --- --- 694

Tripling column length increases peak capacity by 55%. Increasing gradient time by 225% increase peak capacity by 25%

Page 44

Increasing gradient time by 225% increase peak capacity by 25%

Flow Rate Limitations Due to Pressure? Higher Temperature Can Help!

Page 45

Higher Temperature as an Aid to Faster Operation

Higher Temperature:Temperature should always be considered as a parameter during speed optimizationgProvides more rapid mass transfer:• Improves efficiency – enhances resolution• Decreases analysis time – faster separations with no loss in resolution• Decreases analysis time – faster separations with no loss in resolution

Decreases Mobile Phase Viscosity• Lowers backpressure – allows for higher flow rates, faster separations, greater

efficiency and use of sub 2-micron columnsefficiency and use of sub 2-micron columnsCan change selectivity – optimize resolution

Be wary of on-column decomposition• Faster flow rates shorten analyte residence time at elevated temperature and lead

to less decomposition for labile compounds

Page 46

Elevated Temperature Reduces Pressure – Expands Column Choices – Cardiac drugsColumn Choices Cardiac drugs

4.6 x 250 mm, 3.5 µmR = 4 3

Column: SB-C18, As described below Mobile Phase: A: 0.1% TFA, 5% MeCN, (v/v) B: 0.08% TFA, 95% MeCN (v/v)Sample: 0.1 mg/ml of cardiac drugs Temperature: 70°C Flow: 2 mL/min. gradient Detection: 230,16 nm

Very high N column P=221 bar

Rs= 5.6Rs= 4.3 column

min2 4 6 8 10 12 14 16

4.6 x 150 mm, 1.8 µmP=418 barRs= 4.9

Rs= 3.6

min2 4 6 8 10 12 14 16

4 6 x 50 mm 1 8 µm250 mm150 mm50 mm% B

s

R 3 4 4.6 x 50 mm, 1.8 µmP=164 bar

20.0112.014.0112.5

2012460

17.510.53.560

00012.5Rs= 3.4Rs= 2.4

Page 47

min2 4 6 8 10 12 14 16

INCREASED RESOLUTION! When Do You Need It? How Do You Get It?When Do You Need It? How Do You Get It?

• Complex Samples Large No of Peaks in Short Time Frame• Complex Samples—Large No. of Peaks in Short Time Frame

• Closely Related Compounds

• Changes in Bonded Phase Have Not improved Resolution

• Changes in Mobile Phase Have Not Improved Resolution

• Temperature Has Not Helped Change Selectivity

What Is Left That Will Improve Resolution?

Page 48

Reduce Particle Size and Maintain Column LengthIncreased “N” in Isocratic Separation

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 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

Rs = 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 = 14862Rs = 1.37

4.6 x 150, 1.8um490 barN = 28669 Rs = 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

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

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

Page 49

min101

min101

min0 5 10 15 201

min0 5 10 15 201

min101

min101

min0 5 10 15 201

min0 5 10 15 201

1.8u Particles Reveal More Information and Improve Detection and IntegrationImprove Detection 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 50

without loss in sensitivity

1.8u Particle Size Increases Gradient ResolutionIncreased Peak Capacity

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 51

min13 14 15 16 17 18

Particle Size - More Peak Capacity with 1.8 um RRHT Columns - Peptide Map of BSARRHT Columns Peptide Map of BSA

673Peak CapacityColumn used

SB C18 2 1 150 1 8

mAU

40

50

Conditions: Columns: as listed, Mobile Phase: A:0.1% TFA in Water B:0.08% TFA in ACN Gradient: 5% B to 60%B in 25 min. Temperature: 80°C Sample: BSA tryptic digest

StartingPressure380 bar673SB-C18, 2.1x150mm, 1.8µ

10

20

30

35% More peak capacity, more resolution

380 bar

min14.5 15 15.5 16 16.5 17 17.5 18 18.5 19

-10

0

mAU

502SB-C18, 2.1x150mm, 3.5µ40

60

Less peak capacity less resolution

StartingPressure105 barPeak Capacity

0

20

Less peak capacity, less resolution

S ll ti l i h k t k it

Page 52

min15 16 17 18 19

-20 Smaller particle size = sharper peaks = greater peak capacity

Tools

Rapid Resolution Compendium CD-ROM

(Please Ask For A Copy – Pub No 5989-5130EN)

• Application Examples

• Technology Overview

S f C f• 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

Page 53

Agilent HPLC Method TranslatorA Simple to Use Tool to Move Methods to RRHT

Mini-Demo Method Translator

p

Page 54

SummaryShorter 1 8 m col mns allo faster anal sis ith• Shorter 1.8um columns allow faster analysis with same resolving power as longer 5um and 3.5 um columns

• Change column diameter if necessary to allow for lower solvent consumption and higher accessible linear 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• Adjust run time and gradient to allow for the shorter column to match k* for longer column or to increase k* vs. longer column.S d th fl t til fl• Speed up the flow rate until max flow or max pressure or loss of resolution

Page 55

Questions?

Page 56

Appendix

Page 57

So What Bonded Phase Do I Choose – Column Choice vs. Sample Polarity More Retention Loss in High Aqueous MPs

Very Polar NonModerately PolarPola

Wettability with Low or No Organic for RPLC

Very Polar Non Polar

Moderately PolarPolar

Rx-SIL (HILIC)Zorbax SIL (HILIC)Z b NH2 (HILIC)

SB-CNSB-C3

SB – allBonus-RP

SB – allRx-C18E li XDB CNZorbax NH2 (HILIC)

300SB – all300Extend-C18SB-AqSB-CN

Eclipse XDB-CNSB-PhenylBonus-RPSB-C8SB-C18

Rx-C18Eclipse XDB-CNEclipse XDB-PhenylEclipse XDB-C8Eclipse XDB-C18

Eclipse XDB-CNEclipse XDB-Phenyl Eclipse Plus C18, C8Eclipse XDB-C8Eclipse XDB-C18

Bonus-RPSB C18Eclipse Plus C18, C8

Eclipse XDB C18Eclipse Plus C18, C8

pODS ClassicOriginal ODS

RPLC 0-40% organic (except Eclipse Plus

5-40%)

HILIC: 98-50% organic

RPLC 0 40% organic

RPLC 10-70% organic (except Eclipse Plus

5-70%)RPLC: 50-98% organic

NARP: MeOH < ACN < IPA

• These recommendations provide rough guidance on choosing a column based on sample type, with the examples we have seen. They are arranged in a general order of preference.

• Other columns will work in some cases and could provide a better separation

RPLC 0-40% organic )< CH2Cl2 < acetone

Page 58

• Other columns will work in some cases and could provide a better separation, depending on the actual analytes and excipients present in the sample.

Considerations for Method TransferSituation:You have an isocratic method developed for 4.6 mm x 150 mm, 5 um column. Run time is approximately 15 min.Pump: Agilent 1100 quaternaryA t l St d d t lAutosampler: Standard autosamplerTCC: 1100 standardDetector: 1100 DAD, max. data rate 20 Hz

T i l tti PW 0 05 iTypical setting, PW = 0.05 min.Flow Cell: 13 μL, 10 mm path lengthFlow Rate: 1.0 mL/min.Column temp 23ºCColumn temp. 23ºCGoals: Decrease run time and improve throughput (5X, if possible)

Save solvent usage and waste (implies smaller column id or shorter run at higher flow rate)

• Can anything be done to speed up these methods with existing equipment?

What modifications can be made and which are most important?

Page 59

• What modifications can be made and which are most important?

Assessing the Current Method and PerformanceHow does the current method perform?• Method type: assay (several analytes) or impurity method

(> 6 analytes at trace levels)M th d t I ti di t th d• Method type: Isocratic or gradient method

• Critical or limiting resolution? More than one critical pair?• Method parameters

– Column: length ID particle sizeColumn: length, ID, particle size– Column temperature– Type of column heater– Mobile phases used

B k– Backpressure– Injection volume– Injection precision (RSD)

• Instrumental Parameters– Injector type: autosampler, manual, loop size– Delay Volume (system volume for isocratic sep’ns)– Extracolumn Volume

Page 60

Isocratic method on Conventional ColumnTocopherols

Column: ZORBAX Eclipse XDB-C18Mobile Phase: 95% ACN: 5% WaterTemp: 23ºCRRHT

p

Conventional4.6 x 150 mm 5 μm

Flow Rate: 1 mL/minP = 37 bar

Temp: 23 CInjection volume: 1 uL

RRHT4.6 x 50 mm 1.8 μm

Flow Rate: 3 mL/minPressure = 229 bar

P = 37 barmAU

60

80

Rs ~ 4.4Rs ~ 5.2

20

40 1.7 min13.5 min

Sample: Vitamin E – α, β, γ-tocopherols in gel capE li XDB C18 i d fi t h i f th d

min0 2 4 6 8 10 12 140

Page 61

Eclipse XDB-C18 is a good first choice for many methods.

Efficiency Ranking of Various Column Geometries and Typical Backpressuresand Typical Backpressures

This RRHT column Replaces These Longer Columns50 mm, 1.8 μm 150 mm, 5 μm, 100 mm, 3.5 μm

100 mm, 1.8 μm 250 mm, 5 μm

Page 62

Where to begin? Assess Your Current MethodAssess your current method4.6 x 150 mm, 5 μm column

Questions to ask?What is the mobile phase composition?

1.5 mL/minRT last = 14 minutes

What is the current backpressure?Injection Volume?Data Rate/Peak Width?What is your limiting resolution with current method?What size column can deliver the

l ti d?resolution you need?Can your current instrument be used to apply the shorter column with smaller particle size?Which changes in method parameters are necessary and can you get the same or similar performance and results?

Page 63

Tocopherol method translation

4.6 x 150 mm, 5 µm XDB-C18Viscosity of 95:5 ACN/water at 23ºC is ~0.43 cp.

4.6 x 50 mm, 1.8 um RRHT XDB-C18Column length in shorter dimensions with 1.8 µm particles is 4.6 x 50 mm RRHT0.43 cp.

Flow Rate is 1 mL/minBackpressure is 37 barStandard flow cell (13 µL)

µ pAt 1 mL/min expected backpressure is 79 bar + ~10 bar (a/s and flow cell) or ~90 barExpected run time will be 1/3 of 14 minutes or 4.67 minutesStandard flow cell (13 µL)

Standard 0.17 mm tubing throughoutPeak Width required 0.1 min

4.67 minutesTry 3 mL/min for run time of 1/9 of 14 min. or 1.55 min.Predicted pressure is 238 bar

Response Time = 2 sec or Data Rate = 2.5 Hz is adequate

Limiting resolution will be approximately the same (4.4) or 4.4 x SQRT(13043/12077) = 4.2, IF no band broadening due to extracolumn volume or data rate.Standard DAD or MWD at fastest setting (20Standard DAD or MWD at fastest setting (20 Hz) with 0.17 mm id tubing adequate but not optimumChoose 0.12 mm i.d. tubing and 5 µL flow cell for better results

Page 64

for better results

Agilent Method Translator

Page 65

Adjust Flow resist. factor for 37- 5 = 32 bar 13 uL flow cell and

0.17 mm tubing

Effective N hurt by EC vol.

4.67 min run at 1 mL/min

For isocratic runs the 2nd row must be set to same %B as row 1

Page 66

5 uL flow cell and 0.12 mm id tubing

With 5 uL flow cell and 0.12 mm id tubing

Page 67

Adjust % max. pressure until desired flow

rate

Final method conditionsrate

Adjust to 3 mL/min

Click radioClick radio button to

allow % max pressure

adjustment

Page 68

Isocratic method on Conventional ColumnTocopherols

Column: ZORBAX Eclipse XDB-C18Mobile Phase: 95% ACN: 5% WaterTemp: 23ºCRRHT

p

Solvent used 15 mL

Conventional4.6 x 150 mm 5 μm

Flow Rate: 1 mL/minP = 37 bar

Temp: 23 CInjection volume: 1 uL

RRHT4.6 x 50 mm 1.8 μm

Flow Rate: 3 mL/minPressure = 229 bar

P = 37 barmAU

60

80

Rs ~ 4.4Rs ~ 5.2Solvent used 5.1 mL

20

40 1.7 min13.5 min

min0 2 4 6 8 10 12 140

Sample: Vitamin E – α, β, γ-tocopherols in gel capE li XDB C18 i d fi t h i f th d

Page 69

Eclipse XDB-C18 is a good first choice for many methods.

Choosing the flow cell size

Page 70

Flow Cells for RRLC

13µl Standard Flow Cell: For highest sensitivityHigh-demanding quantitative work, e.g. analytical method development, QA/QCy p , Q Q

2µl Micro Flow Cell: For highest resolutionUltra-fast semi-quantitative work, U t a ast se qua t tat e o ,e.g. Screening Experiments, HT LC/MS/UV

5µl Semi-micro Flow Cell: Best compromise of sensitivity and resolution

Dimension Sensitivity* Resolution*

13 µl / 10 mm +++ + p yFor good quantitative and qualitative results, e.g. Screening, HT LC/MS/UV, Early Formulation Studies

* D d l ti l diti d l di i

5 µl / 6 mm ++ ++2 µl / 3 mm + +++

Page 71

* Depends on analytical conditions and column dimension

Peak Width Setting – Response Time – Data Rate and Sensitivity Set at fastest rate and then y

Don‘t use for > 0.15 sec peak width!

increase rate until peak width

> 0.15 sec

> 0.3 sec

> 0.6 sec

Recommended settings in ultra-fast LC with 50% peak width between 0.15 and 0.6 sec

For 50% peak width between 0.6 and 1.2 sec

> 1.2 sec

> 3 sec

> 6 sec

Notes: • Noise level changes ~ proportional to the

square root of the change in data rate.• For optimum selectivity and sensitivity the

Peak Width should not be chosen smaller> 12 sec

> 24 sec

> 51 sec

Peak Width should not be chosen smaller than necessary.

• For 50% peak width between 0.3 and 0.6 seconds Peak Width of > 0.005 min is recommended, which correspondes to 40Hz

Peak Width = Peak Width at 50% Peak Height

data rate.• Only for peaks narrower than 0.3sec at half

height, Peak Width of > 0.0025min (80Hz data rate) should be used.

• For highest sensitivity in ultra fast LC the

Page 72

• For highest sensitivity in ultra-fast LC the slit can be increased to 8 or 16nm.

Detector Data Acquisition Rates – Effects on Peak Width Resolution and Peak Capacity in UFLC

Data Peak Resolution Peak 1.2

1.4

c

60

70

Peak Width, Resolution and Peak Capacity in UFLC

Rate Width Capacity

80 Hz 0.300 2.25 61

40 Hz 0.329 2.05 560.6

0.8

1

Peak

Wid

ths

/ se

c

20

30

40

50

Peak

Cap

acity

Peak Width [s]

Peak Capacity

20 Hz 0.416 1.71 44

10 Hz 0.666 1.17 28

5 Hz 1.236 0.67 16

0.2

0.4

0 20 40 60 80 100Data Rate [Hz]

0

10

80Hz versus 20Hz Data Rate:– 40% Peak Width => +40% Peak Capacity+ 30% Resolution => + 70% Apparent Column Efficiency08

1

1.2

1.4

dths

/ s

ec

1.5

2

2.5lu

tion

Peak Width [s] pp y

80Hz versus 10Hz Data Rate:– 120% Peak Width => +120% Peak Capacity+ 90% Resolution => +260% Apparent Column Efficiency0.2

0.4

0.6

0.8

0 20 40 60 80 100

Peak

Wid

0

0.5

1 Reso

l

Resolution (4,5)

Page 73

0 20 40 60 80 100Data Rate [Hz]

Data Rate and Slit Width Effect on S/N Ratio (DAD and MWD, VWD data rate)(DAD and MWD, VWD data rate)

S/N can be optimized with data rate Slit width can be increased to improve S/N (2 uL and 5 uL cells)

Page 74

to improve S/N (2 uL and 5 uL cells)