the one minute chromatographer - agilent · agilent zorbax rrhd sb-c18, 2.1 x 100mm, 1.8 µm sub 1...

The One Minute

Chromatographer

Agilent Technologies, Inc.

2013

H2O (0.05% Formic acid) / 2-98 % Acetonitrile

T = 80°C

V_i = 0.5 µl

WL = 210 nm

DR = 80 Hz

F = 2 ml/min

P = 1085 bar

Agilent ZORBAX RRHD SB-C18, 2.1 x 100mm, 1.8 µm

Sub 1 Minute Separations with RRHD Columns

Longitudinal

diffusion

Smaller Particles Reduce In–Column Dispersion

Increased Efficiency P

late

Heig

ht

H

Linear Velocity u

Eddy Diffusion

Sum Curve: van-Deemter

Resistance to Mass Transfer

H = A + B/u + C u

Large

Particle

Small

Particle

u opt

H min

Sub 2um Particle

Superficially Porous Particle

Two Advances For Better Mass Transfer

SOLID

CORE

Poroshell 120 Columns for HPLC and UHPLC:

• 80-90% efficiency of sub 2um

• At ~40-50% lower pressure

• 2X efficiency of 3.5um (totally porous)

• A 2.7um particle size

• A 2um frit to reduce clogging

• A 600 bar pressure limit

• The particle has a solid core (1.7um) and

porous outer layer with a 0.5um diffusion

path

July 17, 2013

Confidentiality Label

5

Poroshell 120 columns have:

1.7um

0.5um

0.5um

Poroshell 120 – Several Different Phases

EC-C18

EC-C8

EC-CN

Stablebond C18

Stablebond C 8

Stablebond Aq

Bonus RP

HILIC

Phenyl Hexyl

Van Deemter Redux

7

van Deemter

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0 0.5 1 1.5 2 2.5 3 3.5

Flow rate (mL/min)

HE

TP

(cm

)

Eclipse Plus C18, 3.5 µm

Eclipse Plus C18, 1.8 µm

Poroshell 120 C18, 2.6um

Poroshell 120 C18, 2.6 µm

Poroshell 120 C18, 2.7 µm

High Peak Capacity Maximizes Resolution

with Complex Samples – 1.8u Particles

High Peak Capacity Maximizes Resolution with Complex Samples – Poroshell 120

Smaller Particles Improve

Detection and Integration

4.6 x 150, 1.8μm

490 bar

7 Impurities

All 7 Baseline

Separated!

4.6 x 150, 3.5μm

165 bar

7 Impurities

6 Not Baseline

Separated!

4.6 x 150, 5μm

93 bar

4 Impurities

2 Not Baseline

Separated!

Customer Sample, Impurity Method

What is Sensitivity?

Limit of detection (LOD):

“The lowest concentration, or smallest mass flow, which can be distinguished

from the noise by a certain predefined probability (Signal/Noise).”

Noise

Signal

hSignal = 3(2) x hNoise

Limit of detection (LOD):

Analysis of 15 Analgesic Compounds Same Method for all Three Columns

ZORBAX Eclipse Plus C18, 3 x 100 mm, 3.5 µm

ZORBAX RRHD Eclipse Plus C18, 3 x 100 mm, 1.8 µm

Poroshell 120 EC-C18, 3 x 100 mm, 2.7 µm

Ibuprofen:

PW1/2=0.014

S/N=182

nc=43

Ibuprofen:

PW1/2=0.012

S/N=353

nc=54

Ibuprofen:

PW1/2=0.012

S/N=256

nc=56

2 min

June, 2011

Comparison of 4.6 x 250 mm 5 um to

Poroshell 120 EC-C18 4.6 x 100 mm, 2.7um

min 5 10 15 20 25 30

mAU

0

20

40

60

80

100

9.7

12

11.1

16

11.5

96

12.6

74

15.2

48

16.1

51

16.4

35

20.6

87

23.0

76

29.2

90

min 5 10 15 20 25 30

mAU

0

50

100

150

200

250

1.7

19

2.1

89

2.3

11

2.6

06

3.8

67

4.4

37 4.5

58

5.4

50

5.9

20

7.0

37

325 bar

110 bar

Sulfadiazine,

Sulfathiazole

Sulfapyridine

Sulfamerazine,

Sulfamethazine,

Sulfamethazole,

Sulfamethoxypyridazine,

Sulfachloropyridazine

Sulfamethoxazole,

Sulfadimethoxine

Time %B

0 8

33 33

34 33

Column: Eclipse Plus C18

4.6 x 250mm, 5um

Flow Rate: 1 mL/min

Mobile Phase:

A: 0.1% formic acid in Water

B: 0.1% formic acid in ACN

Time %B

0 8

12 33

13.2 33

Column: 4.6 x 100mm

Poroshell 120 EC-C18,

2.7um

Flow Rate: 1 mL/min

Expand High Speed Chromatograms for True

Comparison to Slower Separation

min 5 10 15 20 25 30

mAU

0

20

40

60

80

100

9.7

12

11.1

16

11.5

96

12.6

74

15.2

48

16.1

51

16.4

35

20.6

87

23.0

76

29.2

90

110 bar

5

mAU

0

50

100

150

200

250

1.7

19

2.1

89

2.3

11

2.6

06

3.8

67

4.4

37 4.5

58

5.4

50

5.9

20

7.0

37

325 bar

min

See conditions previous slide

Rapid Resolution LC Ultra Fast, Resolution Maintained, Sensitivity Improved

Optimized Gradient

Time (min)

1

2

4

5

3

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Column: Rapid Resolution Eclipse XDB-C8,

4.6 x 50 mm, 3.5 mm

Mobile Phase: A: 55% 25 mM Na2HPO4, pH 3

B: 45% MeOH

Gradient: 42 – 90% B in 2 min

Flow Rate: 3 mL/min

Temperature: 35°C

Sample: Cardiac Drugs

1. Diltiazem

2. Dipyridamole

3. Nifedipine

4. Lidoflazine

5. Flunarizine 480 Samples/day

Run Time 1.8 min

Equilibration 1 min

Time

Total Analysis 2.8 min

Time

US. Pharmacopeia 621

Column length: ± 70 %

Column internal diameter: ± 25 %

Column material particle size: Reduction of up to 50 %, no increase

Flow rate: ± 50 %

Injection volume: Changes are allowed as long as system suitability

testing (SST) criteria are met.

Column temperature: ± 10 %

pH of mobile phase: ± 0.2

UV wavelength: no change outside manufacturer specifications

Concentration of salts in buffer: ± 10 %

Composition of mobile phase (adjustment of the minor component is

allowed ±30 % or ± 10 % absolute whichever is smaller)

USP and FDA Method Adjustment Criteria Method Adjustment Criteria for Column Dimensions

Parameter Maximum Specifications Comments/Examples

Column Length ± 70% 250mm 75mm

150mm 50mm

Column Internal Diameter

±25% (FDA ORA-LAB 5.4.5*)

USP – ± 25% Column ID can be adjusted provided

linear velocity is constant**

4.6 mm 3.0 mm (-35%)

4.6 mm 2.1 mm (-54%)

3.0 mm 2.1 mm (-30%)

Flow Rate ±50%

Injection Volume

Reduce as much as needed –

must still meet detection limits and

precision

If you change to a smaller/ shorter

column make the appropriate

change in injection volume

Particle Size Reduce by up to 50%

(can not be increased)

You can change column length

and particle size to keep Rs same

5um 3.5um (-30%)

5um 2.7um (-46%)

*Changed on 2/24/09 - For the current and official copy, go to

http://web.ora.fda.gov/dfs/policies/manuals/default.html

** USP 30 Second Supplement Revisions, PF34(5), in process expected to be final Dec 2009

See Stimuli article in Pharmacopeial Forum 2009; 35(6)

http://web.ora.fda.gov/dfs/policies/manuals/default.html

USP Method-Naproxen

Naproxen 100 mm column

NAPROXEN 50 mm Column

Agilent 1200 Rapid Resolution System – Speed

Conventional LC → UFLC → RRLC

F= 1.20ml/min

T = 40°C

Analysis Time = 11min

Solvent Cons. = 13.2ml

High Resolution:

4.6mm x 150mm 5.0µm

min 0 2 4 6 8 10 12

F = 4.80ml/min

T = 40°C

Analysis Time = 1.05min


High Speed:

4.6mm x 50mm 5.0µm

min 0 0.2 0.4 0.6 0.8 1

F= 1.00ml/min

T = 40°C

Analysis Time = 1.1min


High Speed & Resolution:

2.1mm x 50mm 1.8µm

min 0.2 0.4 0.6 0.8 1 0

Max Speed at T = 95oC

2.1mm x 50mm 1.8um

F= 2.40ml/min

T = 95°C

Analysis Time: 0.4min


PWHH = 197msec

min 0.2 0.4 0.6 0.8 1 0

> 20x faster !

Group/Presentation Title

Agilent Restricted

Month ##, 200X

0 10 20 30 40

Changing Gradient Time to Affect Retention (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 = constant

F = 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

Very Fast LC on Conventional 1100 HPLC

G1379 Degasser

G1311 Quaternary pump

G1313A ALS autosampler

G1316A column compartment

G1314A VWD (standard cell G1314-60086, 10mm, 14uL)

Acetophenone

Diethyl phthalate

Benzophenone

Butyrophenone

Valerophenone

Hexanophenone

Heptanophenone

Octanophenone

The Sample

The Instrument

min0 2 4 6 8 10 12 14 16 18

mAU

0

50

100

150

200

250

VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\070809SBC180003.D)

1.4

74

3.3

23

5.0

64

5.6

51

6.1

14

6.9

64

8.3

37

9.6

90

10.9

82

Conventional Column - 4.6 x 150mm, 5µm, SB-C18

Flow Rate 1.0 ml/min

Injection Volume 15uL

Temperature 30°C

Wavelength 246nm

Sample rate 2.5 Hz

Time (min) % Acetonitrile

0 50

10 90

13.5 90

13.6 50

15 50

Initial Pressure: 69 bar

Final Pressure: 38 bar

Shorten Column and Gradient Time by Same Factor 1/3 Column Length- 1/3 Gradient Time

RRHT Column – 4.6 x 50mm, 1.8µm, SB-C18

min0 1 2 3 4 5 6

mAU

0

20

40

60

80

100

120

140

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

1.1

76

2.0

04

2.2

54

2.4

64

2.8

11

3.3

57

3.8

71

4.3

43



Temperature 30°C

Wavelength 246nm

Sample rate 13.74 Hz


0 50

3.33 90

4.5 90

4.53 50

5 50



Increase Column Flow-Reduce Gradient Time Double Flow (2mL/min) – ½ Gradient Time

RRHT 4.6 x 50mm, 1.8µm, SB-C18

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

20

40

60

80

100

120

140

VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS 2007-08-10 08-22-16\070810SBC180004.D) 0

.587

1.0

20

1.1

46

1.2

60

1.4

37

1.7

15

1.9

70

2.2

01



Temperature 30°C

Wavelength 246nm

Sample rate 13.74 Hz


0 50

1.67 90

2.25 90

2.27 50

3.34 50



• Flow rate vs. Gradient time vs.

Peak capacity

• For small molecules(MW < ~1000)

• Different Column Lengths

• Broken lines are isobar (800 bar)

What Length Column Yields Highest Peak Capacity?

50mm

150mm

100mm

Shorter Columns with Fast Gradients

Yield Higher Peak Capacity

50mm

150mm

100mm

Shorter Gradient (5 min)

Peak Capacity:

• 258 for 50 mm

• 240 for 100 mm

• 221 for 150 mm

Long Gradient (40 min)

Peak Capacity:

• 422 for 50 mm

• 510 for 100 mm

• 525 for 150 mm

Longer Columns with Long Gradient Times

Yield Greater Peak Capacity

50mm

150mm

100mm

Constant Particle Size, Grad Time

More Resolution


Agilent Restricted

Month ##, 200X Page 30

min 0 2 4 6 8 10 12 14 16 18

mAU

0

50

100

150

200

min 0 2 4 6 8 10 12 14 16 18

mAU

0

50

100

150

200

min 0 2 4 6 8 10 12 14 16 18

mAU

0

50

100

150

200

RRHD SB-C18 2.1 x 50 mm, 1.8um

Pmax=366 bar

nc = 424

RRHD SB-C18 2.1 x 100 mm , 1.8um

Pmax=595 bar

nc = 485

RRHD SB-C18 2.1 x 150 mm, 1.8um

Pmax=768 bar

nc = 589

Rs: 2.40

Rs: 1.37

Rs: 0

Poroshell 120, 150mm Columns – HPLC or UHPLC

2 mL/min

min 0 2 4 6 8 10 12 14 16 18

mAU

0

20

40

60

80

100

120

min 0 5 10 15 20 25 30 35

mAU

0

20

40

60

80

100

120

1 mL/min P = 285 bar

P = 538 bar

Conditions: Column: Poroshell 120 EC-C18, 4.6 x 150mm, 2.7um Mobile Phase: Solvent A: Water with 0.1% Formic Acid

Solvent B: Acetonitrile 1200 SL controlled temperature at 25 °C 2 ul flow cell

1. Hydroquinone

2. Resourcinol

3. Catechol

4. Phenol

5. 4-Nitrophenol

6. p-cresol

7. o-cresol

8. 2-Nitrophenol

9. 3,4 di methyl phenol



12. 1-napthol

Gradient: 1mL/min

Time %B

6.0 5%

51 60%

Gradient: 2mL/min

Time %B

3.0 5%

25.5 60%

UHPLC/TOF (1290/6230) Can Identify More

Compounds in Less Time

1 .5

min

Time Composition

0.0 10% ACN

1.5 100% ACN

224 pesticides at 50 pg each

217 ionized & detected in positive

mode

(97%, Find by Formula)

2.1 x 50 mm x 1.8 micron

Eclipse Plus C-18

900 bar

1.5 mL/min

1290 Infinity

TOF fast acquisition rates (20Hz) ensure maximum throughput

Ultrafast LC/MS Analysis for 15 Analyte Subset

1290 Infinity Applications

Peak Width 0.7 sec

RRHD Eclipse Plus C18

2.1x 50 mm, 1.8 um

750 bar

1 minute

Time Composition

0.0 10% ACN

1.5 100% ACN

Ultimate speed on a short column with ballistic gradient

Injection Volume

Conversion

Detector Settings

recommendation

Gradient and

Isocratic Method

Conversion (auto-

detected)

Method Translator Makes Changes Easy

Let Agilent Method Translator Do the Math Basic Mode for Easy Transfer of Conventional Method to RRLC

Original Method New Method

Detailed Output Detailed Input

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

http://www.chem.agilent.com/en-US/Products-Services/Instruments-

Systems/Liquid-Chromatography/pages/1200infinity_cost_calculator.aspx

“RULE OF THUMB”

• Set of Approximations based on chromatographic

behavior and mathematical relationships

• Will deliver nearly the desired goal

• Probably need to be tweaked to deliver best results

What Is Your Current Column Efficiency and

How Fast Do You Want To Run?

Column

Length (mm)

Resolving

Power

N(5 µm)

Resolving

Power

N(3.5 µm)

Resolving

Power

N(1.8 µm)

Typical

Pressure

Bar (1.8 µm)

150 12,500 21,000 32,500 560

100 8,500 14,000 24,000 420

75 6000 10,500 17,000 320

50 4,200 7,000 12,000 210

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

15 N.A. 2,100 2,500 55

Analysis Time

Peak Volume

Analysis

Time*

-33%

-50%

-67%

-80%

-90%

Solvent Usage

* Reduction in analysis time compared to 150 mm column

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

First, Reduce Column Length

5um 1.8um

Reduce column length by factor of 3

Quite often original method will have more resolution than is actually

needed and a reduction by 5 may be possible

3.5 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

Flow Modification - Different Diameter Columns

ml/minmm

mmml/min 21.0

4.6

2.11.0 i.e.

2

2 col.

2

column1

column21 col. Flow

.Diam

Diam.Flow

Decreased Column Volume May Require

Conversion for Injection Volume

2 col.

column1

column21 col. Inj.Vol.

Volume

VolumeInj.Vol.

2 col.column1

column21 col. 4

2.0

4.020 i.e. μl

ml

mlμl

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

Reduce injection volume

4.6 mm 3 mm

Reduction to allow for diameter change

2.1 mm 4.6 mm

= 0.4 x Original

= 0.2 x Original

x Reduction 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



Simple Isocratic Method Transfer for Speed

Step 1

• Reduce Column Length and Particle Size

• Maintain Flow Rate

Step 2 Faster

• Increase Flow Rate

• Pressure too High? Increase Temperature to Lower Pressure


Step 3 Even Faster (Need Instrument Optimization)

• Decrease Column Diameter (2.1mm), Reduce Flow Rate Proportionately

• Reduce Injection Volume Based on Ratio of Column Volumes


Simplified Gradient Method Transfer

Step 1

• Reduce Column Length and Particle Size

• Adjust Gradient Time by same Factor

• Maintain Flow Rate

Step 2 Faster

• Increase Flow and Reduce Gradient Time

• Stop When Reach Flow Limits of Instrument

Step 3 Even Faster

• Decrease Diameter of Column (2.1mm)

• Match Flow to New Column Diameter

• Reduce Injection Volume

• Repeat Step 2 Until Reach 80-90 % Instrument Pressure Limit


Agilent Restricted

Month ##, 200X

Let’s Look at Method Development of These

Compounds

diltiazem

dipyridamole

propranolol pindolol

disopyramide

β-blocker

Anti-arrhythmic

Vasodilator

Ca+ channel blocker

http://en.wikipedia.org/wiki/Image:Dipyridamole.svg

http://en.wikipedia.org/wiki/Image:Propranolol.png

http://en.wikipedia.org/wiki/Image:Pindolol.png

http://en.wikipedia.org/wiki/Image:Disopyramide.svg


Agilent Restricted

Month ##, 200X

Start at Low pH, Adjust Organic

Eclipse Plus C18 Cardiac Drugs with Acetonitrile

2

mAU

0

100

200

300

400

2

mAU

0

25

50

75

100

125

min 2 4 6 8 10 12

mAU

-20

0

20

40

60

Column: ZORBAX RRHT Eclipse Plus C18, 4.6 x 50 mm, 1.8 mm

Mobile Phase: A: 25 mM NaH2PO4 , pH 3.0 B: ACN

Flow Rate: 2.0 mL/min Temperature: 30°C Detection: UV 240 nm

Sample: Cardiac Drugs 1.Pindolol 2. Diisopyridamide 3.Propranolol 4.Dipyridamole 5. Diltiazem

40% ACN

20% ACN

30% ACN

k= 44!!

RRHT Eclipse Plus C18 allows for very fast

optimization of % organic in mobile phase.

Good resolution

Fast analysis

No time wasted To get this k on a 25cm

column at 2 mL/min

would require 1.5 hours

run time!!


Agilent Restricted

Month ##, 200X

Start at Low pH on Eclipse Plus C18

Cardiac Drugs with Methanol, Changing Organic Modifier

2

mAU

0

100

200

300

400

2

mAU

0

25

50

75

100

125

min 2 4 6 8 10 12

mAU

0

20

40

60

80

Column: ZORBAX RRHT Eclipse Plus C18, 4.6 x 50 mm, 1.8 mm

Mobile Phase: A: 25 mM NaH2PO4 , pH 3.0 B: MeOH

Flow Rate: 2.0 mL/min Temperature: 30°C Detection: UV 240 nm

Sample: Cardiac Drugs 1.Pindolol 2. Diisopyridamide 3.Propranolol 4.Diltiazem 5. Dipyridamole

70% MeOH

40% MeOH

50% MeOH

k= 49!!

Adjusting MeOH influences

retention like adjusting ACN

Solvents can be compared

quickly with RRHT columns

MeOH changes selectivity and

gives longer analysis time for

these analytes

• When flow limit of pump is reached

Consider using a smaller i.d. column

• When approaching about 90% pressure limit of instrument

• 540 bar for 1200SL pump

• 360 bar for 1100/1200 pump

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

• When resolution is no longer satisfactory

When to Stop!?

Methanol/water 40C

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

0 20 40 60 80 100 120

%Methanol

Vis

co

sit

y

ACN/water 40C

0.00

0.20

0.40

0.60

0.80

0 20 40 60 80 100 120

%ACN

Vis

co

sit

y

Agilent 1.8u and Poroshell 120 Columns

• Make High Resolution, Fast LC Possible on All Instruments

• Reduce Analysis Time

•Speed Up Method Development

•Improves Methods on HPLC

•Provides Best Benefits on UHPLC

•Coupled with UHPLC Instruments Allow Higher Flow Rates for

Higher Resolution Without Long Run Times

the one minute chromatographer - agilent · agilent zorbax rrhd sb-c18, 2.1 x 100mm, 1.8 µm sub 1...

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