Page 1

Tips On Getting

More From Your

Liquid Chromatography

Carl Griffin

Application Engineer

Advanced Chromatography Media

Page 2

ZORBAX RRHT and

RRHD

POROSHELL 120

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

March 18, 2013

Confidentiality Label

3

Poroshell 120 columns have:

1.7um

0.5um

0.5um

Page 4

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):

Page 5

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

Van Deemter Comparision

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

Page 8

USP Method-Naproxen

Page 10

NAPROXEN 50 mm Column

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 B Vm

k* =

= 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

Page 12

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

Page 13

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

Flow Rate 1.0 ml/min

Injection Volume 5uL

Temperature 30°C

Wavelength 246nm

Sample rate 13.74 Hz

Time (min) % Acetonitrile

0 50

3.33 90

4.5 90

4.53 50

5 50

Initial Pressure: 132 bar

Final Pressure: 74 bar

Page 14

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

Flow Rate 2.0 ml/min

Injection Volume 5uL

Temperature 30°C

Wavelength 246nm

Sample rate 13.74 Hz

Time (min) % Acetonitrile

0 50

1.67 90

2.25 90

2.27 50

3.34 50

Initial Pressure: 266 bar

Final Pressure: 146 bar

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

Page 15

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

Data

Rate

Peak

Width

Resolution Peak

Capacity

80 Hz 0.300 2.25 60

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

min 0.1 0.2 0.3 0.4 0.5 0

80Hz

PW=0.30sec

40Hz

PW=0.33sec

20Hz

PW=0.42sec

10Hz PW=0.67sec

5Hz PW=1.24sec

Sample: Phenones Test Mix

Column: Zorbax SB-C18, 4.6x30, 1.8um

Gradient:: 50-100%ACN in 0.3min

Flow Rate: 5ml/min

80Hz versus 10Hz (20Hz) Data Rate

• Peak Width: – 55% (– 30%)

• Resolution: + 90% (+ 30%)

• Peak Capacity: + 120% (+ 40%)

• App. Column Eff.: + 260% (+ 70%)

Detectors For narrow peaks, high data rates!!! Maintaining Resolution at High Analysis Speed

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

Page 18

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

HPLC Instrument Components

Gradient Delay or Dwell Volume .

Extracolumn Volume

Data Sampling or Acquistion Rate

.

Number of Scans

or points

Effects of Delay Injection Program

Page 21

0 10 20 30 40

Minor Dwell Volume Differences

Can Change Resolution

0 10 20 30 40

VD = 0.43

mL

Column: ZORBAX Rapid Resolution Eclipse

XDB-C8

4.6 x 75 mm, 3.5 µm

Mobile Phase: Gradient, 0 - 100 %B in 52.5 min.

A: 5/95 methanol/ 25 mM phosphate

pH 2.50

B: 80/20 methanol/25 mM phosphate

pH 2.50

Flow Rate: 0.5 mL/min

Temperature: 25°C

Injection: 5 L

Detection: 250 nm

Sample: Mixture of antibiotics and

antidepressants

Upper trace simulates actual run data

entered into DryLab® 3.0 software

Lower trace is simulated chromatogram

for larger VD

VD = 2.0

mL

Effect of Extra-column Volume on a Gradient

Analysis of Alkylphenones

Default 1290, 8.6 µL Extra-column Volume

Optimized 1290, 3.0 µL Extra-column Volume

A: H2O; B: CH3CN

0.4 mL/min

t (min) 0 1.2

%B 25 95

1 µL injection of RRLC Checkout Sample (PN 5188-

6529) spiked w/ 50 µL 2 mg/mL Thiourea in

water/acetonitrile

TCC: ambient

DAD: Sig=254,4nm; Ref=Off

Sample:

1. Thiourea (v0 marker)

2. Acetanilide

3. Acetophenone

4. Propiophenone

5. Butyrophenone

6. Benzophenone

7. Valerophenone

8. Hexanophenone

9. Heptanophenone

10. Octanophenone

Agilent ZORBAX Eclipse Plus RRHD C18

2.1 mm x 50 mm, 1.8 µm, 959757-902

LC Rack System, 5001-3726

0.08 x 220 mm Capillary Tubing

V(σ)0.6 µL Flow Cell

Pmax=320 bar

Rs5,6=1.18

nC=35

Pmax=323 bar

Rs5,6=2.25 91% increase

nC=56 60% increase

Page 22

System – Signal Height System volumes – System dispersion

min 0.5 1 1.5 2 2.5

mAU

0

100

200

350

400

550

600

System dispersion optimized

Peak width 0.018 min

Peak width 0.019 min

Resolution 1.902

min 0.5 1 1.5 2 2.5

mAU

0

100

200

300

400

System dispersion not optimized

Peak width 0.038 min

Peak width 0.037 min

Resolution 0.961

310

mAU

380

mAU

System – Signal height System volumes – Delay volume

Delay volume ~ 700 μL

min 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

100

200

300

400

Delay volume ~ 120 μL

min 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

100

200

300

400

Column: ZORBAX SB-C18 2.1 x 50 mm, 1.8 μm

Flow: 0.42 mL/min

120 mAU

220 mAU

Influence post-column capillary connections

min 0 0.1 0.2 0.3 0.4

mAU

0

20

40

60

80

100

120

140 One bad capillary connection!

min 0 0.1 0.2 0.3 0.4

mAU

0

30

60

90

120

150

180

210 Fixed!

130 mAU

160 mAU

Page 25

What Happens If the Connections Poorly Made ?

Page 26

If Dimension X is too long, leaks will occur

Ferrule cannot seat properly

Mixing Chamber

If Dimension X is too short, a dead-volume,

or mixing chamber, will occur

Wrong … too long

Wrong … too short

X

X

Page 27

Why Filter the Sample?

Extreme Performance Requires Better Sample “Hygiene”

• Prevents blocking of capillaries, frits, and the column inlet

• Results in less wear and tear on the critical moving parts of injection valves

• Results in less downtime of the instrument for repairs

• Produces improved analytical results by removing potentially interfering contamination

A Dirty Sample: Precipitated Plasma Column

Life Test

Page

28

Column: 2.1x50mm, SB-C18

Sample: 1mg/ml plasma + 0.2mg/ml naphthalene in 75/25 ACN/H2O solution (filtered)

Method: flow = 0.8 ml/min; temp = ambient; detector = 210 nm; injection = 0.5 ul

Mobile phases: A – H2O + 0.1% TFA; B – ACN + 0.08% TFA

Gradient table: Time (min) 0 0.2 0.7 1.2 1.3 1.5

B% 20 20 90 90 20 Stop

400

500

600

700

800

900

1000

1100

1200

0 500 1000 1500 2000 2500

Pre

ssu

re (

bar)

Injections

RRHD Plasma Sample test

USCES00002

USCES01074

Sample was plasma, protein precipitated,

centrifuged, filtered through 0.2um filter.

Page 29

In-Line Filters Provide Good Insurance

Against System OverPressure NEW RRLC In-line filter and fitting – max 600 bar

Protect RRHT columns with efficient in-line filter with 0.2 µm pore size frits

Description Part number Porosity

Frit

diameter

Flow

rate

Part number

Replacement

Frits

RRLC In-line

filter, 2.1 mm,

max 600 bar

5067-1551 0.2 µm 2.1 mm<1

mL/min

5067-1555

(10/pk)

RRLC In-line

filter, 3.0 & 4.6

mm, max 600

bar

5067-1553 0.2 µm 4.6 mm1 - 5

mL/min

5067-1562

(10/pk)