Maximising Separation, Resolution and Speed

Paul Barton Product Specialist Consumables Division Agilent Technologies

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06/05/2011

Aim of Analysis Maximum Resolution in Reasonable Time

The objective of chromatography is to provide the maximum resolution in the minimum time reproducibly

Rs = N 4
LC Factors affecting

( 1)

k (k+1)

Column length, particle size Stationary phase chemistry, mobile phase chemistry, temperature

Column length, mobile phase strength, temperature

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Typical Method Development Parameters - Effects of Selectivity, Efficiency, and Retention on Resolution
Selectivity Impacts Resolution Most
7.00

6.00

Change bonded phase Change mobile phase

Typical Method Development Parameters

Plates are easiest to increase

5.00

Resolution

4.00

Increase N Increase Alpha Increase k'

3.00

2.00

1.00

Rs = N/4 ( -1)/
0.00

k/(k+1)
13 14 15

Plates: Alpha: k:

5000 1.10 2.0

10000 1.35 4.5

10

15000 1.60 7.0

11

12

20000 1.85 9.5

16

17

18

19

20

25000 2.1 12.0

21

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Change of pH (pH 2 9) Can Be Used to Optimize Selectivity

pH 2.7 0.1% formic acid/MeCN
1 3 2 5
0 2 4 6 8 min

7 4 6

1. procainamide 2. buspirone 3. pioglitazone 4. eletriptan 5. dipyridamole 6. diltiazem, 7. furosemide

pH 4.8 NH4OAc/MeCN
1 2 7
0 2 4

6 5
8

Selectivity and resolution can change with pH Eclipse Plus can be used with many mobile phases and pHs

4
6

min

pH 7 NaPO4/MeCN

7 3 4 5

Conditions: Column: Eclipse Plus C18 4.6 x 100mm, 5um Gradient: 10 90% in 10 minutes Detection: UV 254 nm

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Start at Low pH, Adjust Organic Eclipse Plus C18 Cardiac Drugs with Acetonitrile
mAU
400 300 200 100 0

Column: ZORBAX RRHT Eclipse Plus C18, 4.6 x 50 mm, 1.8 m Mobile Phase: A: 25 mM NaH2PO4 , pH 3.0 B: ACN Flow Rate: 2.0 mL/min Temperature: 30C Detection: UV 240 nm Sample: Cardiac Drugs 1.Pindolol 2. Diisopyridamide 3.Propranolol 4.Dipyridamole 5. Diltiazem

40% ACN
2

RRHT Eclipse Plus C18 allows for very fast optimization of % organic in mobile phase.
Good resolution Fast analysis No time wasted

mAU 125 100 75 50 25 0

30% ACN
2

mAU 60 40 20 0 -20 2 4 6 8

20% ACN

To get this k on a 25cm column at 2 mL/min would require 1.5 hours run time!!

k= 44!!
min

10

12

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06/05/2011

Start at Low pH on Eclipse Plus C18 RRHT Cardiac Drugs with Methanol, Changing Organic Modifier
mAU 400 300 200 100 0

Column: ZORBAX RRHT Eclipse Plus C18, 4.6 x 50 mm, 1.8 m Mobile Phase: A: 25 mM NaH2PO4 , pH 3.0 B: MeOH Flow Rate: 2.0 mL/min Temperature: 30C Detection: UV 240 nm Sample: Cardiac Drugs 1.Pindolol 2. Diisopyridamide 3.Propranolol 4.Diltiazem 5. Dipyridamole

70% MeOH
2

mAU 125 100 75 50 25 0 2 mAU 80 60 40 20 0 2 4 6

50% MeOH

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

40% MeOH
8 10

k= 49!!
12 min

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Bonus-RP Can Provide Alternative Selectivity to Alkyl Phases

mAU 800 400 0 mAU 800 400 0 0 2 4 6 8 min
1 253 6 4 1
Columns: 4.6 x 150 mm Instrument: HP 1100 Mobile phase: MeOH:0.1% TFA (70:30)* Flow rate: 1 mL/min. Temperature: Ambient DAD1 B, Sig=254,16 Ref=360,60 (F:\2\DATA\TRIAZINE\AZINE046.D) Inj.: 2 L UV: 254 nm Sample: 1. Prometryne 5. Diuron 2. Tebuthion 6. Propanil 3. Atrazine 7. Dacthal 4. Propazine

2
3 4

Bonus-RP
7

Alkyl-C8
7

DAD1 B, Sig=254,16 Ref=360,60 (F:\2\DATA\TRIAZINE\AZINE047.D) is compatible preferred over phosphate, and

*For low pH work, a TFA mobile phase is often with LC/MS.

Silica-based Column Packing with Alkyl/amide Stationary Phase

Si NH O O

Zorbax Rx-SIL Silica Support Surface

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Column Efficiency
Number of theoretical plates, N Theoretical plates per metre, N/m Height equivalent to a theoretical plate:

Hmm = Lmm/N
NB: the smaller H, the more efficient the column
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Van Deemter Equation

C term resistance to mass transfer is improved by Reducing particle size this is the current standard approach Improving diffusion
Theoretical Plate height H The smaller the theoretical plate height H, the better the Resolution! large particle H = A + B/u + C x u Resulting van-Deemter curve Resistance to Mass Transfer Longitudinal Diffusion Eddy Diffusion u opt
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small particle

H min

linear flow u

06/05/2011

Van Deemter Curve : HETP vs. Volumetric Flow Rate

0.0030

0.0025

Column: ZORBAX Eclipse XDB-C18 Dimensions: 4.6 x 50/30mm Eluent: 85:15 ACN:Water Flow Rates: 0.05 5.0 mL/min Temp: 20C Sample: 1.0 L Octanophenone in Eluent

H = A + B/u + Cu

5um

HETP (cm)

0.0020

0.0015

3.5um
0.0010

0.0005

1.8um

0.0000 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Volumetric Flow Rate (mL/min) Smaller particle sizes yield flatter curves, minima shift to higher flow rates
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Resolution
Shorter Columns with Smaller Particles Plates Selectivity Retention

Rs =
N

-1

4
L

k' k'+1
Column Length = N

dp
dp/2

Particle Size
Particle Size

=
=

N
P

To Maintain Rs: e.g.: L/2

Page 12

000990P2.PPT

06/05/2011

Column Length and Particle Size

Column Length (mm) 150 100 75 50 30 15

Column Efficiency N(5 m) 12,500 8,500 6000 4,200 N.A. N.A.

Column Efficiency N(3.5 m) 21,000 14,000 10,500 7,000 4,200 2,100

Column Efficiency N(1.8 m) 35,000 23,250 17,500 12,000 6,500 2,500

Efficiency (N) Analysis Time Peak Volume Solvent Usage

Analysis Time*

-33% -50%

Pressure

-67% -80%
-90%

* Reduction in analysis time compared to 150 mm column; all columns 4.6-mm i.d.
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06/05/2011

Rapid Resolution HT Columns Up to 20X Faster

Up to 20x faster than conventional HPLC
With same or better performance (resolution, precision, sensitivity, carry over) Compliant with strictest regulatory performance requirements
SB-C18 4.6 x 150mm, 5m 1.20ml/min, 40C Analysis Time = 11min RRHT SB-C18 2.1mm x 50mm 1.8m 1.00ml/min, 40C Analysis Time= 1.1min

HPLC, 40C

10

min

RRLC, 40C 10x faster

0 0.2 0.4 0.6 0.8 1 min

RRHT SB-C18 2.1mm x 50mm 1.8m 2.40ml/min, 95C Analysis Time: 0.4min

RRLC, 95C

27x faster
0 0.2 0.4 min 0.6

>20X faster

PW = 197msec

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Tools

Method Translation Software

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06/05/2011

Sub-Two Micron Columns Can Be Used In Two Ways

Fast LC with short, highly efficient columns at conventional and high flow rates High Resolution LC with longer (100mm-150mm) columns and conventional flow rates

There is a trade-off between speed and resolution, but it is often possible to have a mixture of slightly faster analysis with more resolution
This comes at a price :Much higher system backpressure

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06/05/2011

Pressure Equation
viscosity flow length

FL P = 0 2 2 K r dp
specific permeability column radius particle diameter

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06/05/2011

Agilent Has Three Types of Columns for UHPLC

There are 3 column types for UHPLC choice depends on your requirements RRHT columns 1.8um columns for fast LC up to 600 bar

Designed for compatibility with the Agilent RRLC

Compatible with some UHPLCs with pressure limits up to 600 bar Designed for compatibility with the new Agilent 1290 Infinity LC Useable up to 1200 bar Fully UHPLC compatible 600 bar pressure limit for use with HPLC/UHPLC UHPLC type performance but excellent fit for 400 bar LCs

RRHD columns 1.8um columns for up to 1200 bar

Poroshell 120 columns

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06/05/2011

Poroshell 120 Columns for HPLC and UHPLC:

Poroshell 120 columns have:
80-90% efficiency of sub 2um At ~40-50% lower pressure 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

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06/05/2011

Step 1 Make the Solid Core

Cores are a uniform 1.7um size with a smooth surface
RSD of seven batches of cores = 0.04%

This leads to a tight final particle size distribution

Tighter than with totally porous particles

SEM of Cores

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06/05/2011

Comparison of Particle Size Distributions Between Totally Porous and Poroshell 120 Particles
Very narrow particle size distribution reduces the A term of the van Deemter equation (Eddy diffusion)

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06/05/2011

Step 2 Make the Porous Outer Layer

The Agilent process for Poroshell 120 differs from other processes for superficially porous particles. A single step coacervation process is used, rather than a multilayering process.

Coacervation is the process by which small sol particles come together to form the porous particle
Like the finely controlled pore size/particle size process of ZORBAX 1 step reduces variability more reproducible

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Comparison of 4.6 x 250 mm 5 um to Poroshell 120 EC-C18 4.6 x 100 mm, 2.7um
mAU 100 80 60 40 20 0 mAU 250
1.719 7.037

16.151 16.435

9.712

11.116

15.248

5
2.311

10

15

20

23.076

110 bar

20.687

25

29.290

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
11.596

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

Sulfadiazine, Sulfathiazole Sulfapyridine Sulfamerazine, Sulfamethazine, Sulfamethazole, Sulfamethoxypyridazine, Sulfachloropyridazine Sulfamethoxazole, Sulfadimethoxine

12.674

30

min

325 bar
2.606 5.450 5.920 4.437 4.558

200 150 100

50
0

Faster analysis (shorter column higher flow rate) More Sensitive (smaller particle sharpens peak) Better Optimized (faster analysis allows better tweaking) Both run on Agilent 1100 G1315B DAD (under 400 bar) No change in sample prep required, 2 micron frit used on both columns
10 15 20 25 30 min

2.189

3.867

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06/05/2011

Comparison of 4.6 x 250 mm 5 um to Poroshell 120 EC-C18 4.6 x 100 mm, 2.7um
See conditions previous slide
12.674

mAU 100
11.116

11.596

9.712

15.248

60 40 20 0

250 200

325 bar
1.719 2.189

2.311

2.606

4.437 4.558

5.450

150 100

50
0

3.867

5.920

7.037

mAU

10

15

20

23.076

110 bar

20.687

25

29.290

80

16.151 16.435

30

min

min

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06/05/2011

Practical Faster GC Applications with Capillary GC Columns

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06/05/2011

Resolution

N k Rs = 4 k 1
Efficiency Retention Selectivity
Page 26

1
L = Length rc = column radius df = film thickness T = temperature

N=
k =

(gas, L, rc)
(T, df, rc)

(T, phase)

06/05/2011

Stationary Phase - Common Types

Siloxane polymers

Poly(ethylene) glycols

Porous polymers

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Optimizing Selectivity
Match analyte polarity to stationary phase polarity -like dissolves like(oil and water dont mix)

Take advantage of unique interactions between analyte and stationary phase functional groups

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06/05/2011

Start with the Right Phase

DB-1 15m x 0.32mm, 0.25m Oven: 40C for 2 min 40-120C at 5C/min

6 Time (min.)

10

12

14

DB-Wax 15m, 0.32mm, 0.25m Oven: 80-190C at 20C/min

3
Time (min.)

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06/05/2011

Column Length and Efficiency (Theoretical Plates)

Length (m) 15 30 60
0.25 mm ID n/m = 4630 (for k = 5)

N 69,450 138,900 277,800

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Column Length VS Resolution and Retention: Isothermal

R=0.84 2.29 min
R=1.16 4.82 min R=1.68 8.73 min

15 m

30 m
Double the plates, double the time but not double the the resolution

60 m

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DECREASE THE LENGTH

DB-5 30 m 0.53 mm I.D., 0.5 m
1 2

4
3 5

0 0 .5

.5

1 0 .0

1.0

1 0 .5

1.5

2 0 .0

2.0

2 0 .5

2.5

3 0 .0

3.0

3 0 .5

3.5

4 0 .0

4.0

4 0 .5

4.5

5.0

5 0 .0

5 0 .5

5.5

6 0 .0

6.0

6 0 .5

6.5

7 0 .0

7.0

7 0 .5

7.5

8 0 .0

8.0

8 0 .5

8.5

9 0 .0

9.0

DB-5 15 m 0.53 mm I.D., 0.5 m

1 2

1. 2. 3. 4. 5.

Benzene Toluene Ethylbenzene m,p-Xylene o-Xylene

4 3

0 0 .2

0 0 .4

1.0 0.60

0 0 .8

1 0 .0

1 0 .2

1 0 .4

2.0 1.60

1 0 .8

2 0 .0

2 0 .2

2 0 .4

3.0 2.60

2 0 .8

3 0 .0

3 0 .2

3 0 .4

4.0 3.60

3 0 .8

4 0 .0

4 0 .2

4 0 .4

5.0

4 0 .6

4 0 .8

5 0 .0

5 0 .2

5 0 .4

6.0

5 0 .6

5 0 .8

BTEX Carrier: Helium, 36 cm/sec at 40c Oven : 40C for 3 min, 5/min to 100C
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Length Summary

If you Decrease Length: Efficiency Resolution Analysis Time Pressure Cost Decrease Decrease Decrease Decrease Decrease

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06/05/2011

Resolution

N k Rs = 4 k 1
Efficiency Retention Selectivity
Page 34

1
L = Length rc = column radius df = film thickness T = temperature

N=
k =

(gas, L, rc)
(T, df, rc)

(T, phase)

06/05/2011

Different Column I. D.
Equal Phase Ratios
Column : DB-624 30 m, 0.53 mm, 3 m Carrier: Helium, 40 (cm/sec) Oven: 65C Injection: Split Detector: FID

Column :

DB-624 30 m, 0.32 mm, 1.8 m

10 Time (min)

15

20

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06/05/2011

PHASE RATIO ( )
Film Thickness

Column Dimensions

Phase Ratio

30 m x .53 mm x 3.0 m
30 m x .32 mm x 1.8 m KC = k = r 2df

44
44

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06/05/2011

Column Diameter - Inlet Head Pressures (Helium)

I.D (mm) 0.05 0.10 Pressure (psig) 275-400 90-130

0.18
0.20

30-45
25-40

0.25
30 meters Hydrogen pressures x 1/2 0.32 0.45 0.53
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15-25
10-20 3-7 2-4
06/05/2011

Column Diameter and Capacity

I.D. (mm) Capacity (ng)

0.05 0.10 0.18 0.20

Like Polarity Phase/Solute 0.25 m film thickness 0.25 0.32 0.45 0.53
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1-2 6-13 25-55 35-70

80-160 110-220 600-800 1000-2000
06/05/2011

Diameter Summary

If you decrease the inside diameter: Efficiency Resolution Increase Increase

Pressure
Capacity Flow rate

Increase
Decrease Decrease

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06/05/2011

Carrier Gas Considerations Golay Plots

Excessive Diffusion

N2

Optimum Practical Gas Velocity, OPGV 1.5 x uopt

1.00

H (min)

0.75 0.50 0.25

uopt 10 20 30 40 50

Poor Mass Transfer

He H2

60

u (cm/sec)
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Agilent High Efficiency (0.18mm i.d.) Columns

0.18mm i.d. columns provide a good trade-off between faster GC and GC/MS analyses and sample capacity
High efficiency columns permit faster analyses with reasonable backpressure, efficiency and robustness with both helium and hydrogen carrier gas Maintaining the phase ratio simplifies method translation A 30m x 0.25mm x 0.25 m column translates to a 20m x 0.18mm x 0.18 m Internal diameter (mm) 0.18 0.25 0.32
Page 41

Film thickness ( m) 0.18 0.25 0.32

Column Theoretical Length (m) Plates 20 30 30 133200 138900 112800

Plates/m

6660 4630 3760

k = 5

06/05/2011

Agilent Method Translation Software

Easy Options with Method Translation Software

Different Column Dimensions

Switch He to H2 Carrier Gas and Try Faster Velocities

Same Column & Gas Type but Faster Velocities

Combination of all of the above

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Food/Fragrance Method translation

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Food/Fragrance Method translation

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06/05/2011

Spearmint Oil

10.6 min
-16.8 min (0.18 mm, H2 Carrier)

17.7 min
-9.7 min (0.18 mm, He Carrier)

(0.25 mm, He Carrier)

27.4 min

13

18

23

28

Time (min)

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06/05/2011

Spearmint Oil Resolution Check

-16.8 min (0.18 mm, H2 Carrier)

10.6 min

-9.7 min (0.18 mm, He carrier)

17.7 min

(0.25 mm, He Carrier)

27.4 min

13

18

23

28

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06/05/2011

Resolution Maintained

Compounds Sabinene -Pinene -Terpinene p-Cymene Speed Gain

0.25 mm Helium 1.52 1.61 N/A

Compound Resolution
0.18 mm Helium 1.59 1.73 35%

0.18 mm Hydrogen 1.56 1.86 61%

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Questions?

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06/05/2011