Superficially porous particles offer improved

efficiency and performance over similarly sized

traditional totally porous particles. Higher efficiency

leads to improved resolution and possible time

savings with superficially porous particles, hence

their growing popularity for HPLC analyses. Columns

using superficially porous particles are currently

available in a wide variety of particle sizes, pore sizes

and stationary phase chemistries to meet most

analysts’ needs. This work will address method

transfer and scalability from totally porous particles,

as well as among different varieties of superficially

porous particles and when to use which particle and

column configurations. Overcoming common barriers

of method transfer with high efficiency columns,

including instrument configuration, method settings,

and proper connections will also be addressed.

SPP Columns, like Agilent Poroshell 120, have a Lower

Reduced Plate Height and Better C-term than 1.8 µm

TPP Columns

Significant Care is Taken to Ensure the Wide Variety of

Agilent TPP and SPP Columns Provide Similar

Selectivity for Predictable and Easy Method Transfer

and Scaling Selectivity Comparisons with 4.6 x 50 mm Columns, 5-95% CH3CN in 2

min with 0.1% formic acid, 2 mL/min

Selectivity plots, using a simple gradient show highly correlated

selectivity among these TPP and SPP Agilent C18 columns for 140

compounds (including acids, bases and neutrals), with R2>0.99 and slopes

close to 1.

Because of the strongly correlated selectivity, transferring and scaling

methods among these Agilent C18 columns is predictive, often requiring

no further method development.

When High pH Mobile Phases are Necessary, the

Agilent Poroshell HPH-C18 Column can Appreciably

Improve Column Lifetime

Lifetime of SPP columns in phosphate buffer, pH 8, at elevated

temperature. Mobile phase: Premixed 60% 30 mM sodium phosphate

buffer at pH 8 and 40% methanol; Flow rate 0.4 mL/min; UV absorbance

254 nm; 65 °C; Columns: 2.1 x 50 mm, 2.7 µm; Analyte: Naphthalene

The HPH SPP particles are synthesized by chemically modifying the

surface of the silica SPP particles with an organic layer, resulting in 15x

longer lifetime under these stressful chromatographic conditions

(columns are considered dead after a 10% loss of efficiency).

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

0.00 5.00 10.00 15.00 20.00

Red

uced

pla

te h

eigh

t, h

Reduced interstitial velocity, v

van Deemter Plot, k'(naphthalene)=3, 2.1 x 50 mm columns

Transferring and Scaling Methods among a Variety of

Superficially Porous Particle Columns Anne Mack, William Long, Jason Link, Xiaoli Wang, Maureen Joseph, Bill Barber; Agilent Technologies, 2850 Centerville Rd, Wilmington, DE

Introduction

Conclusions

High Efficiency Generated by SPP Columns can

Improve Resolution, Making the Difference between

Separating Analytes or Not

The 5 µm TPP column cannot separate these 8

benzodiazepines; however a 2.7 µm SPP column with the same

selectivity has enough efficiency to drive baseline resolution

for all analytes.

High Efficiency Generated by SPP Columns can also

Save Substantial Time and Solvent, Allowing More

Analyses per Day and Lower Costs per Analysis

The 2.7 µm SPP column gives a more sensitive, faster, better

resolved separation of 10 sulfa drugs, compared to a longer 5

µm TPP column, while only generating 325 bar pressure.

Analysis time is reduced by 73% with the SPP column.

Mobile phase consumption is reduced by 47% with the SPP

column.

Recent Revisions to USP General Chapter 621 Allow

For Method Flexibility with SPP Columns of Various

Particle Sizes

Allowable Column Adjustments per USP General Chapter 621

before and after August 1, 2014

Example: SPP Column Alternatives for USP Naproxen Assay

Additional Allowable Adjustments for Method Parameters

Instrument Dispersion Can Have a Considerable Negative Impact on the Performance of Low Volume, High Efficiency SPP Columns Instrument Dispersion is Improved by 60% (Reduced from 9.7 to 3.9 µL) using Short, Small id Connecting Capillaries to Reduce System Extra-Column Volume

Performance of Isocratic Analyses is Significantly Improved by Minimizing LC System Extra-Column Volume; Efficiency is

Improved up to 65%, Resolution is Improved up to 39% Gradient Performance is also Improved by Minimizing System Extra-Column Volume; Peak Capacity is Improved by 27%, Resolution is Improved by >20%

Spring-Loaded Agilent A-Line Fittings Improve Performance by Ensuring Ideal Connections Every Time a Connection is Made The Agilent A-Line Quick Connect fitting won The Analytical Scientist Innovation Award (TASIA) for its highly innovative approach in successfully simplifying the connection of fittings in UHPLC.

Monitoring of Tailing Factor and Theoretical Plates Number over 200 Reconnections of an A-Line Quick Connect Fitting using a Naphthalene QC Analysis

There is no visible change in chromatographic performance after 200 reconnections; tailing factors and theoretical plates stayed constant within the experimental allowance through the procedure. The spring-loaded A-Line Quick Connect Fitting kept the connection dead-volume free and leak-free after 200 times of reuse.

Method transfer and scaling is predictable and

easy with SPPs Similar selectivities are available on both SPPs and

TPPs SPP’s improved efficiency can increase resolution

and save significant time New USP guidelines allow flexibility with SPP

columns Low volume, high efficiency columns are very

sensitive to instrument dispersion Spring-loaded fittings paired with high efficiency

SPP columns ensure ideal connections

Results and Discussion Results and Discussion

1.8 µm

TPP

2.7 µm

SPP

A 1.26 1.06

B 5.83 3.74

C 0.08 0.05

— ZORBAX Eclipse Plus C18, 2.1 x 50 mm, 1.8 µm (p/n 959757-902) —Poroshell 120 EC-C18, 2.1 x 50 mm, 2.7 µm (p/n 699775-902)

y = 1.0163x - 0.0024

R² = 0.9994

0

0.5

1

1.5

2

2.5

3

0 1 2 3

RT

on 2

.7 µ

m P

oros

hell

12

0 E

C-C

18

(m

in)

RT on 4 µm Poroshell 120 EC-C18

(min)

y = 1.0296x + 0.012

R² = 0.9992

0

0.5

1

1.5

2

2.5

3

0 1 2 3

RT

on 5

µm

ZO

RB

AX

Ecl

ipse

Plu

s C

18

(m

in)

RT on 4 µm Poroshell 120 EC-C18

(min)

y = 1.0459x + 0.0608

R² = 0.9989

0

0.5

1

1.5

2

2.5

3

0 1 2 3

RT

on 2

.7 µ

m P

oros

hell

12

0 E

C-C

18

(m

in)

RT on 2.7 µm Poroshell HPH-C18

(min)

50

60

70

80

90

100

110

0 1000 2000 3000

% In

itial

Eff

icie

ncy

mL Stress Buffer

Silica SPP-C18

HPH SPP-C18

—Poroshell 120 EC-C18 2.1 x 50 mm, 2.7 µm (p/n 699775-902) —Poroshell HPH-C18 2.1 x 50 mm, 1.8 µm (p/n 699775-702)

Parameters for

System Suitability

USP34 USP37-NF32S1

Isocratic/Gradient Isocratic Gradient

Particle Size -50% L/dp: -25% to +50%

or N: -25% to +50%

No Changes allowed

Column Length ±70%

Column Inner

Diameter

Flexible, w/ constant

linear velocity

Flexible, w/ constant linear

velocity

No Changes allowed

Parameters for

System Suitability

USP34 USP37-NF32S1

Isocratic/Gradient Isocratic Gradient

Flow rate Based on column

dimension: F2=F1×[(l2×d2

2)/(l1×d12)]

Additional adjustments:

±50%

Based on dp: F2=F1×[(dc2

2×dp1)/(dc12×dp2)]

Additional adjustments:

±50%, provided N

decreases ≤20%

No Changes allowed

Injection volume May be reduced, as far as

is consistent with

precision and detection

limits;

increase not permitted

May be adjusted, as far as

is consistent with precision

and detection limits

May be adjusted, as far as

is consistent with precision

and detection limits

Column Temp ±10oC ±10oC ±10oC

Mobile phase pH ±0.2 units ±0.2 units ±0.2 units

Salt Concentration within ±10% if the

permitted pH is met

within ±10% if the

permitted pH is met

within ±10% if the

permitted pH is met

Ratio of

Components in

Mobile Phase

Minor component

(≤50%): ±30% relative,

cannot exceed ±10%

absolute; may only adjust

1 component in ternary

mixtures

Minor component (≤50%):

±30% relative, cannot

exceed ±10% absolute;

may only adjust 1

component in ternary

mixtures

No Changes allowed *

* Not specified in 621,

assume no changes are

allowed

Wavelength of UV-

Vis Detector

No changes allowed No changes allowed No changes allowed

min 0.5 1 1.5 2 2.5 3

mAU

0 25 50 75

100 125 150

min 0.5 1 1.5 2 2.5 3 3.5

mAU

0 25 50 75

100 125 150

min 0.5 1 1.5 2 2.5 3 3.5

mAU

0 25 50 75

100 125 150

A: 0.1% formic acid in water, B: 0.1% formic acid in acetonitrile, 2 mL/min, 32%B isocratic, 25C, 254nm, 80 Hz

5 µm ZORBAX Eclipse Plus C18 4.6 x 50 mm (p/n 959946-902)

4 µm Poroshell 120 EC-C18 4.6 x 50 mm (p/n 699970-902)

2.7 µm Poroshell 120 EC-C18 4.6 x 50 mm (p/n 699975-902)

1. alprazolam 2. clonazepam 3. diazepam 4. flunitrazepam 5. lorazepam 6. nitrazepam 7. oxazepam 8. temazepam

5 10 15 20

mAU

0 20 40 60 80

100

5 10 15 20 25

mAU

0 50

100 150 200 250

1. sulfadiazine 2. sulfathiazole 3. sulfapyridine 4. sulfamerazine 5. sulfamethazine 6. sulfamethazole 7. sulfamethoxypyridazine 8. sulfachloropyridazine 9. sulfamethoxazole 10. sulfadimethoxine

5 µm ZORBAX Eclipse Plus C18 4.6 x 250 mm (p/n 959990-902)

2.7 µm Poroshell 120 EC-C18 4.6 x 100 mm (p/n 695975-902) 25

A. 0.1% Formic Acid in H2O, B. CH3CN, 1 or 2 mL/min, 8-33% B in 30 or 8 min, 25 C, 254 nm

min 0 1 2 3 4 5 6 7 8 9

mAU

0 10 20 30 40 50 60

USP Prescribed Column: 5 µm Eclipse Plus C18 4.6 x 150 mm

L/dp=30,000

min 0 1 2 3 4 5 6 7 8 9

mAU

0 10 20 30 40 50 60

System Suitability Method Requirement: N>4000, Rs>11.5

min 0 1 2 3 4 5 6 7 8 9

mAU

0 10 20 30 40 50 60

1 2 3 4 5 6 7

mAU

0 5 10 15 20 25 30

1 2 3 4 5 6 7

mAU

0 5 10 15 20 25 30

4 µm Poroshell 120 EC-C18 4.6 x 150 mm

25%↑L/dp L/dp=37,500

4 µm Poroshell 120 EC-C18 4.6 x 100 mm

17%↓L/dp L/dp=25,000

2.7 µm Poroshell 120 EC-C18 4.6 x 100 mm

23%↑L/dp L/dp=37,037

2.7 µm Poroshell 120 EC-C18 4.6 x 50 mm

38%↓L/dp L/dp=18,518

N=10639

Rs=13.7

N=19054 79%↑N

Rs=16.9

N=13186 24%↑N

Rs=14.2

N=21046 98%↑N

Rs=17.0

N=11281 6%↑N

Rs=12.6

50:49:1 CH3CN:H2O:CH3COOH 1.2 mL/min, All Pressures < 300 bar Sample: Naproxen, Butyrophenone

Binary Pump

Binary Pump

Autosampler

Diode Array Detector

Diode Array Detector

Solvent Tray Solvent Tray

Default 1290 LC Optimized 1290 LC

Default 1290 LC:

Needle Seat Capillary: 0.12 x 100 mm = 1.1 µL ALSTCC Capillary: 0.12 x 340 mm = 3.8 µL

TCCDAD Capillary: 0.12 x 220 mm = 2.5 µL

Flow Cell V(σ)1.0 µL = 2.3 µL

2.1 x 50 mm Column = 172.3 µL

Void Volume of Column = 103.9 µL

Optimized 1290 LC:

Needle Seat Capillary: 0.11 x 100 mm = 0.9 µL ALSTCC Capillary: 0.08 x 220 mm = 1.1 µL

TCCDAD Capillary: 0.08 x 220 mm = 1.1 µL

Flow Cell V(σ)0.6 µL = 0.8 µL

2.1 x 50 mm Column = 172.3 µL

Void Volume of Column = 103.9 µL

Autosampler

Column Compartment

Column Compartment

x102

0 0.2 0.4 0.6 0.8 1

x102

0 0.2 0.4 0.6 0.8 1

0.4 0.8 1 1.4 1.8 2.2 2.6 3 3.4 3.8 4.2

Optimized LC/UV System, 3.9 µL extra column volume, Pmax = 240 bar RRHD Eclipse Plus C18, 1290 Infinity LC

0.4 mL/min, 60% CH3CN

Agilent Publication 5990-9502EN

Default LC/UV System, 9.7 µL extra column volume, Pmax = 230 bar RRHD Eclipse Plus C18, 1290 Infinity LC

0.4 mL/min, 60% CH3CN

Agilent Publication 5990-9502EN

4.6 5 5.4 5.8

Rs5,6=2.36 N4=5450 N8=9714 N9=10068

Rs5,6=2.78 N4=7630 N8=11196 N9=10967

x102

0 0.2 0.4 0.6 0.8 1

x102

0 0.2 0.4 0.6 0.8 1

0.1 0.2 0.3 0.4 0.5 0.6

Optimized LC/UV System, 3.9 µL extra column volume, Pmax = 310 bar RRHD Eclipse Plus C18, 1290 Infinity LC

0.4 mL/min, 25-95% CH3CN in 1.2 min

Agilent Publication 5990-9502EN

Default LC/UV System, 9.7 µL extra column volume, Pmax = 300 bar RRHD Eclipse Plus C18, 1290 Infinity LC

0.4 mL/min, 25-95% CH3CN in 1.2 min

Agilent Publication 5990-9502EN

0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8

Rs5,6=1.77 nC=44.0

Rs5,6=2.25 nC=55.7

A-Line Quick Connect Assembly p/n

A-Line Quick Connect Assy ST

0.12x105mm 5067-5957