column choice for speed and resolution

8/6/2019 Column Choice for Speed and Resolution

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06/05/2011

Maximising Separation, Resolution

and Speed

Paul BartonProduct Specialist

Consumables DivisionAgilent Technologies



06/05/2011

Aim of Analysis – Maximum Resolution inReasonable Time

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

Rs = √N ( – 1) k

4 (k+1)LC Factors affecting

N Column length, particle size

Stationary phase chemistry, mobile phase chemistry, temperature

k Column length, mobile phase strength, temperature



06/05/2011

Typical Method Development Parameters - Effects ofSelectivity, Efficiency, and Retention on Resolution

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

R e s o l u t i o n

Increase NIncrease Alpha

Increase k'

Selectivity Impacts Resolution Most

• Change bonded phase

• Change mobile phase

• Plates are easiest to increase

Rs = N½/4 ( -1)/ k’/(k’+1)

Plates: 5000 10000 15000 20000 25000

Alpha: 1.10 1.35 1.60 1.85 2.1

k’: 2.0 4.5 7.0 9.5 12.0

Typical Method Development Parameters



06/05/2011

Change of pH (pH 2 – 9) Can Be Used to OptimizeSelectivity

1. procainamide2. buspirone

3. pioglitazone4. eletriptan5. dipyridamole6. diltiazem,7. furosemide

min0 2 4 6 8

1

2

34

5

6

7pH 2.7 0.1% formic acid/MeCN

min0 2 4 6 8

1

7 4

26

5 3

pH 4.8 NH4OAc/MeCN

0 2 4 6 8

1

2

3

4 5

67pH 7 NaPO4/MeCN

Selectivity andresolution can

change with pH

Eclipse Plus canbe used with many

mobile phases andpH’s

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



06/05/2011

Start at Low pH, Adjust OrganicEclipse Plus C18 Cardiac Drugs with Acetonitrile

2

mAU

0

100

200

300

400

2

mAU

0

25

50

75

100

125

min2 4 6 8 10 12

mAU

-20

0

20

40

60

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

m

Mobile Phase: A: 25 mM NaH2PO4 , pH 3.0 B: ACNFlow 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 fastoptimization of % organic in mobile phase.

Good resolutionFast analysisNo time wasted To get this k on a 25cm

column at 2 mL/minwould require 1.5 hours

run time!!



06/05/2011

Start at Low pH on Eclipse Plus C18 RRHTCardiac Drugs with Methanol, Changing Organic Modifier

2

mAU

0

100

200

300

400

2

mAU

0

25

50

75

100125

min2 4 6 8 10 12

mAU

0

20

40

60

80

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

Mobile Phase: A: 25 mM NaH2PO4 , pH 3.0 B: MeOHFlow Rate: 2.0 mL/min Temperature: 30°C Detection: UV 240 nmSample: Cardiac Drugs 1.Pindolol 2. Diisopyridamide 3.Propranolol 4.Diltiazem 5. Dipyridamole

70% MeOH

40% MeOH

50% MeOH

k= 49!!

Adjusting MeOH influencesretention like adjusting ACN

Solvents can be comparedquickly with RRHT columnsMeOH changes selectivity andgives longer analysis time forthese analytes


http://slidepdf.com/reader/full/column-choice-for-speed-and-resolution 7/4806/05/2011Page 8

Bonus-RP Can Provide AlternativeSelectivity to Alkyl Phases

mAU

0

400

800 : .

min0 2 4 6 8

mAU

0

400

800 : .

Columns: 4.6 x 150 mmInstrument: HP 1100

Mobile phase: MeOH:0.1% TFA (70:30)*Flow rate: 1 mL/min.Temperature: AmbientInj.: 2 µLUV: 254 nmSample: 1. Prometryne 5. Diuron

2. Tebuthion 6. Propanil3. Atrazine 7. Dacthal4. Propazine

*For low pH work, a TFA mobile phase is oftenpreferred over phosphate, and is compatiblewith LC/MS.

Bonus-RP

Alkyl-C8

7

1

23 4

5 6

7

1

2 5 6

43

Si

O

NH O

Zorbax Rx-SIL Silica Support Surface

-



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

Column Efficiency

06/05/2011Page 9



Van Deemter Equation

T h e o r e t i c a l P l a t e h e i g h t H

linear flow u

Eddy Diffusion

Resulting van-Deemter curve

Resistance to Mass Transfer

H = A + B/u + C x u

large particle small particle

The smaller the theoretical plate height H,

the better the Resolution!

u opt

H min

Longitudinal Diffusion

C term – resistance to mass transfer is improved by•Reducing particle size – this is the current standard approach

•Improving diffusion

Page 10 06/05/2011



Van Deemter Curve : HETP vs. Volumetric Flow

Rate

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Column: ZORBAX Eclipse XDB-C18

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

Flow Rates: 0.05 – 5.0 mL/min

Temp: 20°C

Sample: 1.0 L Octanophenone in Eluent

Volumetric Flow Rate (mL/min)

H E T P

( c m )

Smaller particle sizes yield flatter curves, minima shift to higher flow rates

H = A + B/ u + Cu

5um

3.5um

1.8um

Page 11 06/05/2011


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Resolution

Shorter Columns with Smaller Particles

Rs = N

4

k'

k'+1

Plates Selectivity Retention

LN

dp

To Maintain Rs:

e.g.: L/2 dp/2000990P2.PPT

Column Length = N

Particle Size = N

Particle Size = P

-1

06/05/2011



Column Length and Particle Size

ColumnLength

(mm)

ColumnEfficiency

N(5 µm)

ColumnEfficiency

N(3.5 µm)

ColumnEfficiency

N(1.8 µm)

150 12,500 21,000 35,000

100 8,500 14,000 23,250

75 6000 10,500 17,500

50 4,200 7,000 12,000

30 N.A. 4,200 6,500

15 N.A. 2,100 2,500

Efficiency

(N)

Pressure

AnalysisTime

Peak

Volume

AnalysisTime*

-

-33%

-50%

-67%

-80%

-90%

SolventUsage

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

06/05/2011Page 13



Rapid Resolution HT Columns – Up to 20X Faster

RRHT SB-C182.1mm x 50mm 1.8µm

1.00ml/min, 40°C

Analysis Time= 1.1min

RRLC, 40°C

10x faster

min0.2 0.4 0.6 0.8 10

HPLC, 40°CSB-C184.6 x 150mm, 5µm

1.20ml/min, 40°C

Analysis Time = 11min min0 2 4 6 8 10

0.2 0.60 0.4 min

RRHT SB-C182.1mm x 50mm 1.8µm

2.40ml/min, 95°C

Analysis Time: 0.4min

RRLC, 95°C

27x faster

PW = 197msec

>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

06/05/2011Page 14


http://slidepdf.com/reader/full/column-choice-for-speed-and-resolution 14/48Page 15

Tools

Method Translation Software

06/05/2011



Sub-Two Micron Columns Can Be Used In TwoWays

• Fast LC with short, highly efficient columns at conventional and

high flow rates

• High Resolution LC with longer (100mm-150mm) columns andconventional flow rates

There is a trade-off between speed and resolution, but it is oftenpossible to have a mixture of slightly faster analysis with moreresolution

This comes at a price :-

Much higher system backpressure

06/05/2011Page 16



Pressure Equation

viscosity flow length

specificpermeability

columnradius

particlediameter

ΔP =ηFL

K0πr2dp2

06/05/2011Page 17



Agilent Has Three Types of Columns for UHPLC

There are 3 column types for UHPLC – choice depends on yourrequirements

RRHT columns – 1.8um columns for fast LC up to 600 bar

• Designed for compatibility with the Agilent RRLC

• Compatible with some UHPLC’s with pressure limits up to 600 bar

RRHD columns – 1.8um columns for up to 1200 bar

• Designed for compatibility with the new Agilent 1290 Infinity LC

• Useable up to 1200 bar

• Fully UHPLC compatible

Poroshell 120 columns• 600 bar pressure limit for use with HPLC/UHPLC

• UHPLC type performance but excellent fit for 400 bar LC’s

06/05/2011Page 18



Poroshell 120 Columns for HPLC and UHPLC:

80-90% efficiency of sub 2umAt ~40-50% lower pressureA 2.7um particle sizeA 2um frit to reduce clogging

A 600 bar pressure limit

The particle has a solid core (1.7um) andporous outer layer with a 0.5umdiffusion path

Poroshell 120 columns have:

06/05/2011Page 19



Step 1 – Make the Solid Core

Cores are a uniform 1.7um sizewith a smooth surface

RSD of seven batches of cores= 0.04%

This leads to a tight final particlesize distribution

Tighter than with totally porousparticles

SEM of Cores

06/05/2011Page 20



Comparison of Particle Size Distributions BetweenTotally Porous and Poroshell 120 Particles

Very narrow particle sizedistribution reduces the Aterm of the van Deemterequation (Eddy diffusion)

06/05/2011Page 21



Step 2 – Make the Porous Outer Layer

The Agilent process for Poroshell 120 differs

from other processes for superficiallyporous particles.

A single step coacervation process is used,

rather than a multilayering process.

Coacervation is the process by which smallsol particles come together to form theporous particle

Like the finely controlled pore size/particle

size process of ZORBAX1 step reduces variability – more reproducible

06/05/2011Page 22



Comparison of 4.6 x 250 mm 5 um to Poroshell 120EC-C18 4.6 x 100 mm, 2.7um

min5 10 15 20 25 30

mAU

0

20

40

60

80

100

9 . 7

1 2

1 1 . 1

1 6

1 1 . 5

9 6

1 2 . 6

7 4

1 5 . 2

4 8

1 6 . 1

5 1

1 6 . 4

3 5

2 0 . 6

8 7

2 3 . 0

7 6

2 9 . 2

9 0

min5 10 15 20 25 30

mAU

0

50

100

150

200

250

1 . 7

1 9

2 . 1

8 9

2 . 3

1 1

2 . 6

0 6

3 . 8

6 7

4 . 4

3 7 4 . 5

5 8

5 . 4

5 0

5 . 9

2 0

7 . 0

3 7

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 onboth columns

325 bar

110 bar

Sulfadiazine,SulfathiazoleSulfapyridine

Sulfamerazine,Sulfamethazine,Sulfamethazole,Sulfamethoxypyridazine,SulfachloropyridazineSulfamethoxazole,Sulfadimethoxine

Time %B0 8

33 3334 33Column: Eclipse Plus C184.6 x 250mm, 5umFlow Rate: 1 mL/min

Mobile Phase:A: 0.1% formic acid in WaterB: 0.1% formic acid in ACN

Time %B0 812 33

13.2 33Column: 4.6 x 100mmPoroshell 120 EC-C18,2.7umFlow Rate: 1 mL/min

06/05/2011Page 23



Comparison of 4.6 x 250 mm 5 um to Poroshell 120EC-C18 4.6 x 100 mm, 2.7um

min5 10 15 20 25 30

mAU

0

20

40

60

80

100

9 . 7

1 2

1 1 . 1

1 6

1 1 . 5

9 6

1 2 . 6

7 4

1 5 . 2

4 8

1 6 . 1

5 1

1 6 . 4

3 5

2 0 . 6

8 7

2 3 . 0

7 6

2 9 . 2

9 0

110 bar

5

mAU

0

50

100

150

200

250

1 . 7

1 9

2 . 1

8 9

2 . 3

1 1

2 . 6

0 6

3 . 8

6 7

4 . 4

3 7 4

. 5 5 8

5 . 4

5 0

5 . 9

2 0

7 . 0

3 7

325 bar

min

See conditions previous slide

06/05/2011Page 24



Page 25

Practical Faster GC Applications withCapillary GC Columns

06/05/2011



Page 26

Resolution

R N k

k

s =

4 1

1

Efficiency

Retention

Selectivity

L = Length

rc = column radius

df = film thickness

T = temperature

N = (gas, L, rc)

k = (T, df, rc)

= (T, phase)

06/05/2011



Page 27

Stationary Phase - Common Types

Siloxane polymers

Poly(ethylene) glycols

Porous polymers

06/05/2011



Page 28

Optimizing Selectivity

Match analyte polarity to stationary phase polarity

-like dissolves like(oil and water don’t mix)

Take advantage of unique interactions between analyte andstationary phase functional groups

06/05/2011



Page 29

Start with the Right Phase

Time (min.)

0 2 4 6 8 10 12 14

Time (min.)

DB-115m x 0.32mm, 0.25µm

Oven:

40°C for 2 min

40-120°C at 5°C/min

0 1 2 3 4 5

DB-Wax

15m, 0.32mm, 0.25µm

Oven:

80-190°C at 20°C/min

06/05/2011



Page 30

Column Length and Efficiency (Theoretical Plates)

0.25 mm IDn/m = 4630 (for k = 5)

Length (m) N

15 69,450

30 138,900

60 277,800

06/05/2011



Page 31

Column Length VS Resolution and Retention:Isothermal

Double the plates, double the timebut not double the the resolution

15 m 60 m30 m

R=0.84

2.29 min

R=1.68

8.73 min

R=1.16

4.82 min

06/05/2011



Page 32

BTEX

Carrier: Helium, 36 cm/sec at 40°c

Oven : 40°C for 3 min, 5°/min to 100°C

1. Benzene

2. Toluene

3. Ethylbenzene

4. m,p-Xylene

5. o-Xylene

.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0

. . . . . . . . . . . . . . . . . .

1

2 3

4

5

DB-5

30 m

0.53 mm I.D., 0.5 µm

DB-5

15 m

0.53 mm I.D., 0.5 µm

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.0 2.0 3.0 4.0 5.0

1 2 3

4

5

6.0

DECREASE THE LENGTH

06/05/2011



Page 33

Length Summary

If you Decrease Length:

Efficiency Decrease

Resolution Decrease

Analysis Time Decrease

Pressure Decrease

Cost Decrease

06/05/2011



Page 34

Resolution

N = (gas, L, rc)

k = (T, df, rc)

= (T, phase)

R N k

k s =

4 1

1

Efficiency

Retention

Selectivity

L = Length

rc = column radius

df = film thickness

T = temperature

06/05/2011



Page 35

Different Column I. D.Equal Phase Ratios

Time (min)

0 5 10 15 20

Carrier:Oven: 65°CInjection: SplitDetector: FID

Column: DB-62430 m, 0.32 mm, 1.8 m

Helium,40(cm/sec)

Column: DB-62430 m, 0.53 mm, 3 m

06/05/2011



Page 36

PHASE RATIO ( )

Film Thickness

Column Dimensions Phase Ratio β

30 m x .53 mm x 3.0 m 44

30 m x .32 mm x 1.8 m 44

KC = k β

r2df

β =

06/05/2011



Page 37

Column Diameter - Inlet Head Pressures (Helium)

30 metersHydrogen pressures x 1/2

I.D (mm) Pressure (psig)

0.10 90-130

0.20 25-40

0.25 15-25

0.32 10-20

0.45 3-7

0.53 2-4

0.18 30-45

0.05 275-400

06/05/2011



Page 38

Column Diameter and Capacity

Like PolarityPhase/Solute0.25 µm film thickness

I.D. (mm) Capacity (ng)

0.05 1-2

0.18 25-55

0.20 35-70

0.25 80-160

0.32 110-220

0.53 1000-2000

0.45 600-800

0.10 6-13

06/05/2011



Page 39

Diameter Summary

If you decrease the inside diameter:

Efficiency Increase

Resolution Increase

Pressure Increase

Capacity Decrease

Flow rate Decrease

06/05/2011



Page 40

Excessive Diffusion

Poor Mass Transfer

Carrier Gas Considerations

Golay Plots

10 20 30 40 50 60

0.25

0.50

0.75

1.00

u (cm/sec)

H

( m i n )

He

N2

H2

uopt

Optimum PracticalGas Velocity,

OPGV ≈ 1.5 x uopt

06/05/2011

A il Hi h Effi i (0 18 i d ) C l



Agilent High Efficiency (0.18mm i.d.) Columns

• 0.18mm i.d. columns provide a good trade-off between faster GC andGC/MS analyses and sample capacity

• High efficiency columns permit faster analyses with reasonablebackpressure, efficiency and robustness with both helium and hydrogen carriergas

• 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

Internaldiameter

(mm)

Filmthickness

( m)

ColumnLength (m)

TheoreticalPlates

Plates/m

0.18 0.18 20 133200 6660

0.25 0.25 30 138900 4630

0.32 0.32 30 112800 3760

k’ = 5

06/05/2011Page 41



Agilent Method Translation Software

E O ti ith M th d T l ti S ft



Page 43

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

06/05/2011

F d/F M th d t l ti



Page 44

Food/Fragrance – Method translation

06/05/2011

F d/F M th d t l ti



Page 45

Food/Fragrance – Method translation

06/05/2011

S i t Oil



Page 46

Spearmint Oil

Δ -9.7 min (0.18 mm, He Carrier)

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

17.7 min

3 8 13 18 23 28

(0.25 mm, HeCarrier)

Time (min)

27.4 min

06/05/2011

S i t Oil R l ti Ch k



Page 47

10.6 min

17.7 min

27.4 min

(0.25 mm, He Carrier)

Δ -9.7 min (0.18 mm, He carrier)

Δ -16.8 min (0.18 mm, H2 Carrier)

Spearmint Oil – Resolution Check

06/05/2011

Resolution Maintained



Page 48

Resolution Maintained

Compound ResolutionCompounds 0.25 mm

Helium

0.18 mm

Helium

0.18 mm

Hydrogen

Sabinene

β-Pinene 1.52 1.59 1.56

α-Terpinene

p-Cymene1.61 1.73 1.86

Speed Gain N/A 35% 61%

06/05/2011

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column choice for speed and resolution

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