speakers moderator - · pdf filehigh efficiency and the van deemter equation 3. sub 2 m silica...

Speakers

Mike Swartz Research Director Synomics Pharmaceutical Services

Scott Fletcher Technical Development Leader CHROMacademy

Moderator

Dave Walsh Editor In Chief LCGC Magazine

Rapidly Becoming Confused! Sub 2µm and Superficially Porous Stationary HPLC Phases

1. Setting the Scene on High

Efficiency HPLC

2. High Efficiency and the Van

Deemter equation

3. Sub 2 m Silica Particles

4. Downsides and Instrument

Requirements

5. Superficially Porous Particles

as an alternative

6. Translating Methods from

traditional particles

Aims & Objectives

Courtesy of Agilent Technologies, Santa Clara, CA

1. So what exactly is high

efficiency HPLC?

2. Increase in the number of

theoretical plates (N) per

separation

3. N for 100 x 4.6mm x 5 m

column = 5000 to 8000

4. N for 100 x 4.6mm x 1.8 m

column = 100,000+

5. Can achieve:

o Sharper peaks

o Higher peak capacity

o Higher resolution

o Higher throughput

Setting the Scene – High Efficiency HPLC

2

2/1

2

54.516W

t

W

tN r

b

r

H = L / N

Van Deemter Equation (1)

Van Deemter Equation (2)

1. Analyte molecules move

through different paths

within the column

packing material

2. Larger differences in

path-length with larger

particles and larger

particle size distributions

3. Eddy diffusion can be

minimized by:

oWell packed columns

oReduced diameter

packing materials

oNarrow particle size

distribution packing

Van Deemter (A Term) - Eddy Diffusion

Movie 1

1. Analytes diffuse in every

direction in the column

2. Longer analyte retention

leads to increased (axial)

diffusion

3. Longitudinal diffusion

can be exacerbated by:

oTubing length / i.d.

oNon-zero dead volume

fittings

oIncorrect column nuts and

ferrules

oHigh volume detector flow

cells (vs’ sensitivity

requirements)

B Term – Longitudinal Diffusion

Movie 2

1. Diffusion into and out of

the pore structure is a

fixed rate process

2. This results in a ‘spread’

of elution times

3. Mass Transfer effects

can be minimized by:

oSmaller diameter

particles

oLow mobile phase flow

rates

oIncreasing column

temperature

Van Deemter (C Term) – Mass Transfer

Movie 3

Van Deemter (C Term) – Mass Transfer

Movie 4

1. Most people will be operating at the ‘practical optimum’ linear

velocity

2. In this region the shape and position of the curve is dictated

by the C-Term and the A-term

3. However, the effect from the B-Term must be negligible – see

latter section on instrumentation

Increasing Efficiency – How?

The two benefits associated with high efficiency separations are;

Increased Efficiency - Improved Resolution

1. Increasing Resolution

Driving toward a reduced Van Deemter Minimum.

The increased efficiency can be utilised to improve resolution

of complex mixtures. This is usually achieved by reducing the

diameter of the particles but maintaining column dimensions


The two benefits associated with high efficiency separations are;

Increasing Efficiency – Faster Analysis

2. Increasing Sample Throughput / Reducing Analysis Time

In this case the composite van Deemter curve remains much

flatter post minimum – usually in conjunction with a reduction

in plate height. This is usually achieved by a reduction in

particle diameter, column dimensions and an increase in flow

and temperature

Courtesy of Agilent Technologies,

Santa Clara, CA

Increasing Throughput – Increasing Temperature

1. By increasing the separation temperature the C-Term is

reduced leading to a flatter post-minimum curve and faster

eluent linear velocity without compromising efficiency

2. This is due to the kinetic energy of the analytes being

increased as they diffuse in and out of the pores and the

viscosity of the ‘stagnant’ mobile phase in the pores is reduced

3. Care should be taken

to ensure that the

analyte does not

thermally degrade or

that peak co-elution or

inversion takes place

Increasing Efficiency – Reducing Particle Diameter

1. By reducing the particle diameter (size) we are reducing the

contributions from both the A-Term and the C-Term

2. Not only does this reduction lead to flatter curve post

minimum, it also leads to an overall reduction in plate height

1. The ‘price’ associated with employing reduced diameter

particles is an increase in operating back pressure

2. Even when increasing the separation temperature, the effect

of reducing particle diameter, especially if combined with a

reduction in column id and an increase in flow, is often

sufficient to necessitate the use of a high pressure UHPLC

instrument

Increasing Efficiency – Reducing Particle Diameter

3. Sub 2µm packed columns

use 0.5µm frits in order to

prevent loss of ‘fines’.

Frits of this size are very

prone to blocking unless

judicious sample preparation

is employed

UHPLC Instrumentation 1. We are experiencing an unheralded time when so many

manufacturers are launching new high pressure

instrumentation platforms

UHPLC Instrumentation

There are four key areas that differentiate UHPLC instruments

from standard HPLC instruments

1. Pump Adaptations

The traditional pressure limit for conventional HPLC

systems is 400bar (~6000psi)

UHPLC systems can operate at pressures exceeding

1000bar (14,500psi) and some UHPLC systems permit the

use of high flow rates at these pressures

In UHPLC pumps the pistons and pistons seals have

been redesigned to withstand the pressures, with some

systems incorporate silica carbide pistons in place of the

traditional synthetic sapphire


2. Gradient Dwell Reduction VD

The dwell volume is the total volume of the system from

where mobile phase mixing occurs to the analytical column

It usually exhibited as an isocratic hold at the start of a run

In order to achieve fast separations ‘ballistic’ gradients are

employed


3. Extra Column Volume ReductionVEC

“The total volume contributed by all system components and

capillary tubing from sample injection to detection, that are

not directly involved with the separation process”

The analyte band will diffuse in the mobile phase as it passes

through the system – van Deemter B-Term

Extra columns volume is the predominant factor in loss of

efficiency when using narrow internal diameter columns

Peak dispersion can be calculated by the Aris-Taylor equation;


3. Extra Column Volume ReductionVEC - Continued

From the previous equation we can conclude that the amount

the analyte band disperses is proportional to the length of

capillary tubing

However it is inversely proportional to the internal

diameter of the of the capillary tubing raised to the 4th power!


3. Extra Column Volume ReductionVEC - Continued

Many manufacturers employ

differently designed column end

fittings

It is essential to match the correct

ferrule type and depth with column

end fitting

Failure to do so can lead to a leak

from an unsecured connection or

an increase in extra column volume

resulting from a void

UHPLC Instrumentation 3. Improvements in Detector Speed

Data acquisitions rates are generally measured in

Hz – measurements per second

UHPLC peaks are much more efficient and narrow and

require faster acquisition speeds

Often mistaken for physical peak dispersion!

Courtesy of Agilent Technologies,

Santa Clara, CA

Increasing Efficiency: An alternative - SPS 1. Superficially Porous Silica particles (SPS)

were first developed over 40 years ago

2. They were used for the analysis of larger molecules

that conventionally generated inefficient, broad peaks

3. Consist of solid core or bead surrounded by a shallow

porous layer

Increasing Efficiency: An alternative - SPS

1. Modern SPS particles have;

• An overall diameter of 2.6 – 3.0µm

• A porous layer of 0.35 – 0.50µm

• Pore widths of 80 – 120Å (8 -12nm)

2. They generate near identical van Deemter curves to fully

porous sub 2µm particles

Increasing Efficiency: An alternative - SPS

1. Whilst they can achieve efficiencies

similar to 1.8µm fully porous particles,

they produce much lower operating

back pressures due to their larger

overall particle diameters

Sample (in peak elution order):

1. saccharin

2. caffeine

3. p-hydroxybenzoic acid

4. aspartame

5. dehydroacetic acid

6. benzoic acid


Increasing Efficiency: An alternative - SPS 1. Historically the benefits of

using SPS particles for the

analysis of larger molecules

was an increased efficiency

due to a reduction in the C-

Term

2. Smaller molecules (≤600Da)

do not suffer the same

hindered mass transfer effects

3. The increased efficiency is due to a much narrower particle

size distribution thus generating a very homogenously, densely

packed column – reduction in A-Term

4. SPS columns utilise standard 2µm frits, thus negating the

propensity to blockage

Converting HPLC methods to UHPLC Instruments

1. In order to successfully translate (geometrically scale) a

current HPLC method onto UHPLC instrument it is

important to follow some important rules

2. Flow rate (isocratic methods only), injection volume and

gradient times all need to be translated to ensure

satisfactory chromatography is achieved and enhanced

resolution and / or reduction in analysis time is realised

3. The resultant pressure can also be predicted

4. ‘Calculators’ available from most instrument / column

manufacturers and suppliers websites but good to have

knowledge of calculations used


1. We are going to translate the isocratic method as detailed

below on to a 50 x 2.1mm, 1.9µm UHPLC column

Conditions Column 1 Conditions Column 2

Column length (mm): 1L = 150

Column internal diameter (mm): 1CD = 4.6

Column particle size (μm): 1Pd = 5.0

Flow rate (mL/min): 1F = 1.0

Injection volume (μL): 1iV = 10

Pressure (bar): 1P

= 54

Gradient conditions Time %A %B

Initial Conditions 1gt = 0 95 5

Step 2 (initial hold) 1gt = 10 40 60

Step 3 1gt = 11 5 95




Flow rate (mL/min): 2F = ?

Injection volume (μL): 2iV = ?

Pressure (bar): 2P

= ?



Step 2 (initial hold) 2gt = ? 40 60

Step 3 2gt = ? 5 95


1. Flow rate conversion for Isocratic method

2

1

2

12

C

C

D

DFF

F2 - new flow rate, F1 - current flow rate,

DC2 - new column id, DC1 - current column id

2

26.4

1.2min/0.1

mm

mmmLF

F2 = 0.21mL / min


2. Injection Volume conversion

Vi2 = 0.7µL

1

2

2

1

212

L

L

D

DVV

C

Cii

mm

mm

mm

mmLVi

150

50

6.4

1.210

2

2

Vi2 - new injection volume, Vi1 - current injection volume,


L2 - new column length, L1 - current column length


3. Pressure conversion

P2 - new pressure, P1 is the current pressure,

dP1 particle diameter of current column, dP2 - new column,

DC2 new column id, DC1 - current column id


P2 = 126bar

11

22

2

22

1112

FL

FL

Dd

DdPP

CP

CP

min/0.1150

min/21.050

1.29.1

6.40.554

2

2mLmm

mLmm

mmm

mmmbarP


4. Converted Method







Pressure (bar): 1P

= 54




Step 3 1gt = 11 5 95





Injection volume (μL): 2iV = 0.7

Pressure (bar): 2P

= 126




Step 3 2gt = ? 5 95


1. We are going to translate the gradient method as detailed

below on to a 50 x 2.1mm, 1.9µm UHPLC column







Pressure (bar): 1P

= 54




Step 3 1gt = 11 5 95




Flow rate (mL/min): 2F = ?

Injection volume (μL): 2iV = ?

Pressure (bar): 2P

= ?




Step 3 2gt = ? 5 95

The flow rate remains the same as it is the change in mobile

phase composition (gradient) that dictates retention


1. Injection Volume conversion

Vi2 - new injection volume, Vi1 - current injection volume



Vi2 = 0.7µL

1

2

2

1

212

L

L

D

DVV

C

Cii

mm

mm

mm

mmLVi

150

50

6.4

1.210

2

2


2a. Gradient Segment Conversion

DC1 - column id, L1 - column length

W - column porosity (~68% or 0.68)

VM1 = 1695µL

or

VM1 = 1.7mL

VM1 – current column void volume

WLD

V C

M 1

2

1

12

68.01502

6.42

1 mmmm

VM


2b. Gradient Segment Conversion

DC2 - column id, L2 is column length

W - column porosity (~68% or 0.68)

VM2 = 117.8µL

or

VM1 = 0.12mL

VM2 – new column void volume

WLD

V CM 2

2

22

2

68.0502

1.22

2 mmmm

VM


2c. Gradient Segment Conversion

tG - gradient segment time, F is the flow rate,

S - shape selectivity value (5 in most cases),

ΔФ - mobile phase composition change, VM - column volume

Therefore

In order to maintain selectivity, the average retention factor, k*,

must be maintained

M

G

VS

Ftk *

2

2

1

11

22

2

11 M

G

M

G

VS

Ft

VS

Ft



Therefore;

The flow, shape, selectivity and change in mobile phase

composition will remain the same.

2

2

1

1

M

G

M

G

V

t

V

t

1

212

x

M

MGG

V

Vtt



Initial gradient segment translation.

1

212

x

M

MGG

V

Vtt

mL

mLtG

7.1

12.0min102

min71.02Gt


2d. Gradient Segment Conversion

Final gradient segment translation.

1

212

x

M

MGG

V

Vtt

mL

mLtG

7.1

12.0min112

min78.02Gt


3. Pressure conversion

P2 - new pressure, P1 - current pressure,

dP1- current particle diameter, dP2 - new particle diameter,



P2 = 600bar

11

22

2

22

1112

FL

FL

Dd

DdPP

CP

CP

min/0.1150

min/0.150

1.29.1

6.40.554

2

2mLmm

mLmm

mmm

mmmbarP


4. Converted Method







Pressure (bar): 1P

= 54




Step 3 1gt = 11 5 95





Injection volume (μL): 2iV = 0.7

Pressure (bar): 2P

= 600



Step 2 (initial hold) 2gt = 0.71 40 60

Step 3 2gt = 0.78 5 95

1. High efficiency HPLC has real benefits in terms of speed

and resolution

2. The Van Deemter equation and its band broadening

concepts help us to understand what the advantages are

3. Advances in silica particle technology help us realise these

benefits – Sub 2 m and Superficially Porous Materials

4. There are trade-offs in all approaches and instrument

requirements need to be considered

5. Translating methods from ‘traditional’ technologies needs

careful consideration

6. The benefits of high efficiency are available to everyone!

Summary

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