An Efficient Approach to Column Selection in HPLC Method ...

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Nov 12, 2013 (3 years and 1 month ago)

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An Efficient Approach to Column
Selection in HPLC Method
Development

Craig S. Young and Raymond J. Weigand


Alltech Associates, Inc.

2051 Waukegan Road • Deerfield, IL 60015

Phone: 1
-
800
-
ALLTECH • Web Site: www.alltechweb.com

Introduction

Common Mistakes in Method Development:




Inadequate Formulation of Method Goals




Little Knowledge of Chemistry of Analyte Mixture




Use of the First Reversed Phase C18 Column Available




Trial and Error with Different Columns and Mobile Phases


These Mistakes Result In:




Laborious, Time
-
consuming Development Projects




Methods that Fail to Meet the Needs of the Analyst

HPLC Method Development
-

A Proposed Procedure

At Your Desk




Define your knowledge of the sample




Define your goals for the separation method




Choose the columns to be considered


In the Laboratory




Choose the initial mobile phase chemistry




Choose the detector type and starting parameters




Evaluate the potential columns for the sample




Optimize the separation conditions (isocratic or gradient) for the



chosen column




Validate the method for release to routine laboratories

Choosing the Appropriate HPLC Column Should
Be Based Both Upon Knowledge of the Sample
and Goals for the Separation



Benefits of this Approach Include:




Small initial time investment




Big time savings in the HPLC laboratory




More “informed” approach to column selection




More efficient than “trial and error” approach

Knowledge of the Sample Influences the Choice of
Column Bonded Phase Characteristics

Knowledge of the Sample




Structure of sample components?




Number of compounds present?




Sample matrix?




pK
a

values of sample components?




Concentration range?




Molecular weight range?




Solubility?




Other pertinent data?

Column Chemistry

(bonded phase, bonding type,
endcapping, carbon load)

Goals for the Separation Influence the Choice of
Column Particle Physical Characteristics

Goals for the Separation




Max. resolution of all components?




Partial resolution?




Fast analysis?




Economy (low solvent usage)?




Column stability and lifetime?




Preparative method?




High sensitivity?




Other goals?

Column Physics

(particle bed dimensions,
particle shape, particle
size, surface area, pore
size)

Column Selection Chart


Method
Goals


High Efficiency

High Capacity

Low
Backpressure

High Resolution

High Sample
Loadability

Suitable for MW
>2000

High Stability

High Sensitivity

Fast Analysis

Low Mobile
Phase
Consumption

Stability at pH
Extremes

Fast Eqilibration

Default Column

(Good for most

Applications)

Particle Size


small (3µm)









medium (5µm)




large (10µm)







Column Length


short (30mm)




















medium (150mm)




long (300mm)








Column ID


narrow (2.1mm)

















medium (4.6mm)




wide (22.5mm)









Surface Area


low (200m
2
/g)


















high (300m
2
/g)











Pore Size


small (60Å)









medium (100Å)




large (300Å)










Carbon Load


low (3%)













medium (10%)




high (20%)











Bonding Type



monomeric






polymeric




















Particle Shape


spherical














irregular





Choosing the Bonded Phase

Draw the molecular structures for all known components of the
mixture. Identify the two compounds whose structures are the
most similar.


e.g.:

O
O
H
O
O
H
O
H
H
O
O
H
O
O
O
Prednisolone

Prednisone

For these two molecules, circle the structural features that differ. It
is these differences that should be exploited to optimize the
separation.


e.g.:

Choosing the Bonded Phase

Prednisolone

Prednisone

O
O
H
O
O
H
O
H
H
O
O
H
O
O
O
Choosing the Bonded Phase

Use the results of the structural comparison to select a bonded
phase showing optimal selectivity for these two molecules. In this
case consider using a silica column (no bonded phase) for its ability
to retain polar solutes through hydrogen bonding.

O
O
H
O
O
H
O
H
H
O
O
H
O
O
O
Prednisolone

Prednisone

Functional Group Polarity Comparisons

Polarity

Functional Group

Structure

Bonding Types

Intermolecular Forces Displayed


Low

Methylene


s

䱯湤潮L



偨敮祬



s


p

䱯湤潮



Hali摥



s

䱯湤潮Ⱐ䑩灯pe
-
䑩灯pe



E瑨敲



s

䱯湤潮Ⱐ䑩灯pe
-
䑩灯p攬eH
-
扯湤b湧



Ni瑲t



s


p


䱯湤潮Ⱐ䑩灯pe
-
䑩灯p攬eH
-
扯湤b湧



E獴敲



s


p


䱯湤潮Ⱐ䑩灯pe
-
䑩灯p攬eH
-
扯湤b湧



Al摥桹摥



s


p


䱯湤潮Ⱐ䑩灯pe
-
䑩灯p攬eH
-
扯湤b湧



䭥瑯湥



s


p


䱯湤潮Ⱐ䑩灯pe
-
䑩灯p攬eH
-
扯湤b湧



A浩湯



s


p


䱯湤潮Ⱐ䑩灯pe
-
䑩灯p攬eH
-
扯湤b湧ⰠA捩c
-
扡獥 捨敭楳瑲t



Hydroxyl



s

䱯L摯測 䑩灯le
-
䑩灯l攬 H
-
扯湤i湧


High

Carboxylic Acid



s


p


䱯湤潮Ⱐ䑩灯pe
-
䑩灯p攬eH
-
扯湤b湧ⰠA捩c
-
扡獥 捨敭楳瑲t

R
(CH
2
)
2
R
R
F, Cl, Br, I
R
O
R
N
+
O
O
-
R
O
O
R
R
O
R
H
O
R
R
R
N
H
2
R
O
H
O
O
H
R
Choosing the Bonded Phase

C18 or Octadecylsilane (ODS)

Very nonpolar

-

Retention is based on London (dispersion)
interactions with hydrophobic compounds.

Example Alltech Phase: Alltima


C18

Examples of bonded phases used for HPLC packing media:

Si
R
R
(CH
2
)
17
CH
3
Choosing the Bonded Phase

Phenyl

Nonpolar

-

Retention is a mixed mechanism of hydrophobic and
p

-

p

interactions.

Example Alltech Phase: Platinum


Phenyl

Si
R
R
(CH
2
)
3
C
C
C
C
C
C
H
H
H
H
H
Choosing the Bonded Phase

Cyanopropyl

Intermediate polarity

-

Retention is a mixed mechanism of
hydrophobic, dipole
-
dipole, and
p

-

p

interactions.

Example Alltech Phase: Alltima


CN


Si
R
R
(CH
2
)
3
C
N
Choosing the Bonded Phase

Each bonded phase has unique selectivity for certain sample types.


As a practical example, to separate toluene and ethyl benzene:




Note a difference of one
-
CH
2
-

unit




Choose a C18 bonded phase for retention by hydrophobicity




Maximize hydrophobic selectivity with a high silica surface area,


high carbon load material like Alltima C18

Toluene

Ethyl Benzene

Choosing the Particle Physical Characteristics

Use the Column Selection Chart




Use “default” column as starting point




Match up method goals with individual particle physical characteristics




Change only those particle parameters that affect the method goals




Recognize the “optimum” column as a possible compromise



Example:



Sample Type: hydrophobic compounds


Method Goal: highest resolution



Choosing the Particle Physical Characteristics

Column Selection Chart



Default Column

Optimum Column†


Column Bed Dimensions


150 x 4.6mm

250 x 4.6mm

Particle Size


5µm

3* or 5µm

Surface Area


200m
2
/g

>200m
2
/g

Pore Size


100Å

100Å

Carbon Load


10%

16
-

20%

Bonding Type


Monomeric

Mono
-

or Polymeric

Base Material


Silica

Silica

Particle Shape


Spherical

Spherical


* mobile phase backpressure may be excessive







Optimum Column: Alltima C18

, 5µm, 250 x 4.6mm (Part No. 88056)


*Note that the choice may represent a compromise. Here, the “optimum” column for resolution sacrifices speed.


Example:



Sample Type: hydrophobic compounds


Method Goal: highest resolution



Choosing the Particle Physical Characteristics

Column Dimensions





Length and internal diameter of packing bed


Particle Shape




Spherical or irregular


Particle Size





The average particle diameter, typically 3
-
20µm


Surface Area





Sum of particle outer surface and interior pore surface, in m
2
/gram

Pore Size





Average size of pores or cavities in particles, ranging from



60
-
10,000Å


Bonding Type





Monomeric
-

single
-
point attachment of bonded phase molecule




Polymeric
-

multi
-
point attachment of bonded phase molecule


Carbon Load





Amount of bonded phase attached to base material, expressed as


%C


Endcapping





“Capping” of exposed silanols with short hydrocarbon chains after


the primary bonding step

Choosing the Particle Physical Characteristics

Column Dimensions

Effect on chromatography


Column Dimension




Short

(30
-
50mm)
-

short run times, low backpressure




Long

(250
-
300mm)
-

higher resolution, long run times




Narrow

(


2.1mm)
-

higher detector sensitivity




Wide

(10
-
22mm)
-

high sample loading

Particle Shape

Effect on chromatography


Spherical particles offer reduced back pressures and longer column
life when using viscous mobile phases like 50:50 MeOH:H
2
O.





Particle Size

Effect on chromatography


Smaller particles offer higher efficiency, but also cause higher
backpressure. Choose 3µm particles for resolving complex, multi
-
component samples. Otherwise, choose 5 or 10µm packings.

Surface Area

Effect on chromatography


High surface area generally provides greater retention, capacity
and resolution for separating complex, multi
-
component samples.
Low surface area packings generally equilibrate quickly,
especially important in gradient analyses.

High surface area silicas are used in Alltech’s Alltima

,
Adsorbospherel
®

HS, and Adsorbosphere
®

UHS packings. Low
surface area silicas are used in Alltech’s Platinum

,
Econosphere

, and Brava


packings.

Pore Size

Effect on chromatography


Larger pores allow larger solute molecules to be retained longer
through maximum exposure to the surface area of the particles.
Choose a pore size of 150Å or less for sample MW


2000.
Choose a pore size of 300Å or greater for sample MW > 2000.



Bonding Type

Effect on chromatography


Monomeric bonding offers increased mass transfer rates, higher
column efficiency, and faster column equilibration.


Si
R
R
(CH
2
)
17
CH
3
Si
C
H
3
C
H
3
(CH
2
)
17
CH
3
X
O
H
+
monomeric
bonding
Si
C
H
3
X
(CH
2
)
17
CH
3
X
+
polymeric
bonding
O
H
O
H
O
O
Si
C
H
3
(CH
2
)
17
CH
3
Polymeric bonding offers increased column stability, particularly
when highly aqueous mobile phases are used. Polymeric bonding
also enables the column to accept higher sample loading.

Carbon Load

Effect on chromatography


Higher carbon loads generally offer greater resolution and longer
run times. Low carbon loads shorten run times and many show a
different selectivity, as in Alltech’s Platinum line of packings.

Endcapping

Effect on chromatography


Endcapping reduces peak
-
tailing of polar solutes that interact
excessively with the otherwise exposed, mostly acidic silanols. Non
-
endcapped packings provide a different selectivity than do
endcapped packings, especially for such polar samples.

Alltech’s Platinum


EPS packings are non
-
endcapped to offer
enhanced polar selectivity.



Conclusion

In this approach to HPLC column selection, the bonded phase chemistry
of the column is chosen on the basis of an analysis of the sample
component structures. The physics of the column is chosen according
to an analysis of the goals for the separation method. This approach
succeeds in predicting unique, optimum bonded phase chemistries and
particle bed physical characteristics that are likely to meet the goals for
the separation method.

Column Selection Example #1

What goals do I have for the method?

Maximum resolution of all components?





Best Peak Shape for difficult samples?







Fast analysis?










Economy (low solvent consumption)?







Column stability
-
long lifetime?







Purify one or more unknown components for characterization?



High sample loadability?



High sensitivity?







…Other (High Sample Throughput
--
Quick Equilibration)







Number of compounds present






4


Sample matrix








--


pKa values of compounds?






--


UV spectral information about compounds?



UV
-
254

Concentration range of compounds





--


Molecular weight range of compounds




94
-

323

What do I know about the sample?

Column Selection Example #1

Structures of Compounds












O
H
(CH2)
5
CH
3
N
(CH
2
)
3
CH
3
Phenol
3-Butylpyridine
Anthracene
3-Hexylanthracene
Column Selection Example #1

Which two sample components have the most similar structures?

Draw them, then circle the structural differences between them.

















Normal phase


silica

NH
2

CN

Reversed phase


C18

C8

Ph

CN



(CH2)
5
CH
3
Anthracene

3
-
Hexylanthracene

Note: The structural difference
between these two compounds is the
hydrophobic hexyl side chain. This
suggests a non
-
polar C18 or C8
column would interact with this area of
difference to help provide separation
of these two compounds.

Recommended bonded phase (silica based materials only)


mark one

Column Selection Example #1

Column physical characteristics


use Column Selection Chart and
Method Goals







Default Column Ideal Column
Column bed dimensions (mm)

150 x 4.6


100 x 2.1

Particle Size (
µ
m)



5



5


Surface area (m
2
/g)


200



<
200


Pore Size (Å)




100



100


Carbon Load (%)



10



10


Bonding type




Monomeric


Monomeric


Particle shape




spherical


spherical



Available packing alternatives meeting the above criteria:


Packing Base


Particle
Particle

Carbon


Pore


Surface

Bonding

End
-



Material

Shape

Size


Load

Size Area Type

cap’d





(
µ
m)


(%)


(Å)


(m
2
/g)





silica


Sph.

3, 5, 10

12

80

220

Mono.

Yes



silica

Sph.

3, 5

8.5

145

185

Mono.

Yes



silica

Sph.

3, 5, 10

10

80

200

Mono.

Yes



silica

Sph.

3, 5, 10

6

100

200

Mono.

Yes






Column of choice:
Brava BDS C18, 100x2.1, 5
µ
m (Spherical , 185m
2
/g,
monomeric)



Column Selection Example #1

Increased Sensitivity,

Low Solvent Consumption,

Fast Analysis

Quick Equilibration

Good balance of efficiency

& backpressure

Reduced

backpressure

Best Peak Shape

Allsphere ODS
-
2



Brava BDS C18




Econosphere C18




Platinum C18

Column Selection Example #2

What goals do I have for the method?

Maximum resolution of all components?






Partial resolution, resolving only select components?



Fast analysis?



Economy (low solvent consumption)?



Column stability
-
long lifetime?









Purify one or more unknown components for characterization?



High sample loadability?



High sensitivity?









…Other



Number of compounds present






6+


Sample matrix








--


pKa values of compounds?






--


UV spectral information about compounds?




UV
-
254

Concentration range of compounds





--


Molecular weight range of compounds




349
-

645

What do I know about the sample?

Column Selection Example #2

Structures of Compounds












N
N
H
S
O
O
O
N
H
2
O
H
H
H
N
N
N
H
S
S
N
O
O
O
O
O
N
H
2
O
H
O
H
H
N
S
N
S
N
N
N
N
N
N
H
O
S
O
O
O
H
H
H
N
N
H
S
N
O
O
O
O
O
O
N
H
2
O
H
O
H
H
N
N
N
N
N
N
N
H
S
N
H
N
S
O
O
O
O
O
O
O
H
O
H
H
H
N
S
N
H
O
S
O
O
O
O
O
H
H
H
Column Selection Example #2

Which two sample components have the most similar structures?

Draw them, then circle the structural differences between them.







Notes: both structures very






polar, with amine and pi bond






functions
--
a RP CN column may





give good separation by mixed
-






mode retention of hydrophobic,





CN
---
H
---
NR
2
hydrogen bonding





and
p
-
p

interactions with double





bonds.




Normal phase


silica

NH
2

CN

Reversed phase


C18

C8

Ph

CN



N
N
H
S
O
O
O
N
H
2
O
H
H
H
N
S
N
H
O
S
O
O
O
O
O
H
H
H
Recommended bonded phase (silica based materials only)


mark one

Column Selection Example #2

Column physical characteristics


use Column Selection Chart and
Method Goals







Default Column Ideal Column
Column bed dimensions (mm)

150 x 4.6

250 x 2.1

Particle Size (
µ
m)



5


5


Surface area (m
2
/g)


200


200 +


Pore Size (Å)




100


Not critical


Carbon Load (%)



10


--


Bonding type




Monomeric

Polymeric


Particle shape




spherical

spherical



Column Selection Example #2

Available packing alternatives meeting the above criteria:


Packing

Base

Particle

Particle

Carbon

Pore

Surface

Bonding

End
-


Material

Shape

Size

Load

Size

Area

Type

cap’d





(
µ
m)


(%)


(Å)


(m
2
/g)




Adsorbosil CN

silica

Irreg.

5, 10

--

60

450

Poly.

Yes


Alltima CN

silica

Sph.

3, 5

--

100

350

Poly.

Yes


Allsphere CN

silica

Sph.

3, 5, 10

--

80

220

Mono.

No


Platinum CN

silica

Sph.

3, 5, 10

--

100

200

Mono.

No






Column of choice:
Alltima CN, 250 x 2.1 , 5
µ
m ( Spherical , 350 m
2
/g ,
polymeric)



High resolution,

High sensitivity

High res.

Good balance of efficiency

& backpressure

Reduced

backpressure

Robust