Polymeric media for high pressure/high performance liquid chromatography. Simulated-Monolith. Non leaching. Heat resistant. Narrow Bore.

1. Why Polymerics as HPLC
Media and Why Simulated
Monoliths™?
HYPHENATION WITH MASS SPECTROMETER AND ITS REQUIREMENTS ARE SOME OF
THE REASONS AND THERE ARE MORE…
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2. About polymeric media
 Polymeric packings media made of polystyrene divinylbenzene are inherently and
uniformly hydrophobic and do not need additional ligands for reversed phase
liquid chromatography.
 They are used in separations of synthetic oligomers, synthetic polymer
compositional analysis, biomolecules, peptides, proteins and oligonucleotides
and can stand harsh conditions.
 Properly manufactured as hard gel polymerics, they are mechanically stable and
unlike silica they are not brittle and do not leach.
 They can be operated at high temperatures as well as high pH’s.
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3. About polymeric media
 There is no phase collapse issue in the absence of organic solvents and the media
can tolerate complete aqueous phase at any pH.
 Polystyrene divinyl benzene media do not have the disadvantage of heavy metal
ion issues on the surface as silica does.
 They can be used in narrow bore columns of 2 mm ID with high performances in
order to minimize solvent consumption as well as increase sensitivity.
 Or, in microbore columns of 1 mm ID to accommodate the hyphenation with
mass spectrometer as detectors.
 Due to their higher retention compared to Silica they operate at higher organics
making them more compatible with mass spectrometers.
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4. About polymeric media
 Using polymerics in capillary columns of 0.3 mm or less almost exclusively with
mass spectrometer as detectors, the leaching becomes the primary focal point to
consider.
 In summary, polymerics offer an alternative to silica to operate at very low or very
high pH’s. They can stand much higher temperatures than silica and they are a
good fit for small columns and can accommodate the LC/MS hyphenation.
 They also comply with USP L21 designation.
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5. About Monoliths/Simulated Monoliths™
 It is well established that Monolith columns have a life cycle that exceeds that of
particulate columns.
 The column lifetime along with its reproducibility are therefore important factors
when making comparison and assessing the purchase value.
 The mechanical robustness, the ability to withstand the full pH ranges from 1 to
14 as well as the endurance to elevated temperatures sets monolith columns
apart.
 Added to the previous properties one should understand that there is no risk of
fracturing the bed in case of unintended fall when dealing with polymerics. The
monolith columns can tolerate what Silica columns can’t.
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6. About Monoliths/Simulated Monoliths™
 There is also the issue of “wall effects”. Monoliths have the
tendency to pull away from the sides of the column in which they
were encased. As a result the mobile phase finds its path around
the stationary phase decreasing resolution. Although advanced
technology has reduced this phenomenon through column
construction it has yet to address it completely.
 Simulated Monoliths™ however has been the answer to both the
“wall effects” as well as the column size limitations monolithic
columns have had thus far. An added obstacle for their use in
process scale.
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7.  Traditional chromatography media are prepared as
porous particles that are packed in columns.
Most of their adsorptive surface area resides within
shallow, dead-end and slow diffusive pores.
 In order to maximize resolution the size of the beads
needs to be reduced. This results in increased column
pressure drop often times close to the upper limits of
traditional HPLC instruments and therefore the need
for Ultra High Performance Liquid Chromatography
(UHPLC) instrumentations.
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About Monoliths/Simulated Monoliths™

8.  Monoliths on the other hand are single rod stationary
phases that are cast in columns.
 They are homogeneous and consist of fully
interconnected network of pores that give direct
access to solutes that flow through it.
 There is a substantial difference in pressure drop when
compared to similar resolution particulate packings.
 Let’s now take a closer look at polymeric monoliths
and their major advantages over traditional
particulates stationary phases.
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About Monoliths/Simulated Monoliths™

9. The architecture of Monoliths/Simulated
Monoliths™
 Rather than the slow diffusion, the fast convection is the
operating mode in monoliths.
 As it shows the adsorptive surface is directly accessible to solutes
during their transition in the column.
 Dynamic binding capacity with monoliths is no longer affected by
the increase in linear velocity.
 Nor is the resolution affected by large solutes such as bulky
proteins or viral particles.
 The convective channels can accommodate large molecules such
as murine leukemia virus with an average size of 1,500 Angstrom.
 The pore size issue becomes obsolete as there is no more
diffusive pore in monoliths or Simulated Monoliths™.
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10. Comparison with porous particles
 The slow diffusive pore of particulate media needs
additional time to elute.
 Appropriate pore size are needed for each category and
size solutes.
 As in size exclusion which is a slow process as well, the
larger molecules are not retained by smaller pores.
 As we will see further on, smaller molecules will be trapped
and are manifested as peak tailing.
 The larger solutes on the other hand will elute faster and
will show as peak fronting.
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11. THE FOCUS OF THIS PRESENTATION:
We will be focusing on Narrow Bore columns as they are geared towards low
waste generation as well as lower level detection using mass spectroscopy as the
preferred and practical method of detection as well as the more efficient method
in identifying solutes in minute quantities in a mixture.
As the detection level decreases the leaching becomes more cumbersome and
needs more attention.
When using mass spectrometer as detector, the end user has to avoid additives
and solvents that are not compatible with the instrument.
The more organic the better the detection when using electrospray.

12. Assessing a column upon
receipt.
 The end user should assess the column upon receipt in order to
monitor its performance during time.
A “Report” or a “Test Certificate” is usually provided to the end user
along with the column.
 For reversed phase polymerics the good practice is to use a small
molecule in un-retained conditions to be able to measure the void
volume and deduct the “Linear Flow” at any particular “Volumetric
Flow” by dividing the column length by the void volume.
 The pressure drop of a column with known particle sizes would
allow to estimate the particle size of another column with unknown
particle size. In the case of monolith it would allow to find out the
equivalent particle size for that column.

13. In the present case a STYROS® 2R narrow bore column of 150 mm was
run at 0.2 ml/min with ACN:H2O 95:5 to provide a peak at 1.974 minutes
with a pressure drop of 26 bars.
21 3 40
This was compared to a column of known particle sizes
(3µm) and the back pressure was measured under the
same conditions to be 77 bars.
Using the pressure equation one can deduct the
particle size equivalent of STYROS® 2R Simulated
Monolith™ column:
This column provides the higher performance of less
than 3µm particles with a pressure drop of bead sizes of
over 5µm as a Simulated Monolith™ column.
Assessing a column upon
receipt.

14. The tailing of a column is indicative of diffusive pores delaying the elution of the
small eluent (acetone) used in this case.
Although it will not affect the end results if the column does not
leach and is used in tandem with a mass spectrometer.
The new generation of mass spectrometers are forgiving and
would not be affected with such tailing.
The back pressure however is an inconvenience so is the specificity
of pore sizes that restricts the universal use of the media.
Simulated Monoliths™ columns do not have such restrictions.
The size reduction of columns to microbore or capillaries as an
inlet is also limited due to the high pressure drop of the media.
The peak elutes at 2.071 minutes with the column running at 77
bars of back pressure.
21 3 40
Assessing a column upon
receipt.

15. A 2.1x50 mm STYROS® 2R Simulated
Monolith™ column separates 4 Parabens in
5 minutes at a flow rate of 0.2 ml/min. That
is 4,000 cm/hr. of linear flow rate .
The pressure is only 36 bars at the start of a
gradient of 75 to 100 % MeOH in 5 minutes.
It is monitored at 254 nm.
To improve the separation further and remain
with the Narrow Bore format, a longer column
of 150 mm could be used without any back
pressure limitation.
Application: Separation of 4 Parabens on a 2.1x50 mm
STYROS® 2R column
MethylParaben
EthylParaben
PropylParaben
ButylParaben
2 3 41 5 6
75% MeOH
100 % MeOH

16. The volumetric flow is now 0.6 ml/min that is a linear
flow of >12,000 cm/hr. based on the column void
volume.
The column pressure drop is 227 bar at the start of the
gradient and drops further with the increase of
organics.
This pressure drop is below the pressure limit of
traditional HPLC instruments and there is still more
room for column length.
Despite the high linear flow rate the separation has
improved by adding additional length.
The separation is run on an Agilent 1290 at 40ºC and
254 nm. The sample is from Supelco: part # 48270-U.
Application: Separation of 4 Parabens on a STYROS® 2R
2.1 x 150 mm column.
21 3 4 5 6
MethylParaben
EthylParaben
PropylParaben
ButylParaben
75% MeOH
100 % MeOH

17. A mixture of 7 components from Supelco (part #
47271) can be separated at a volumetric flow
rate of 1 ml/min or >20,000 cm/hr. using a
2.1x50 mm STYROS® 2R column.
The back pressure is only 200 bar at the start of
the gradient.
The temperature is set at 40⁰ C using an Agilent
1290 Infinity.
The gradient is from 50 to 100% ACN in 5
minutes and monitored at 254 nm.
21 3 4 5 60
50%
Acetonitrile
100 % Acetonitrile
MethylParaben
EthylParaben
PropylParaben
ButylParaben
HeptylParaben
Phenol
Uracil
Application: Separation of 7 components on a 2.1x50 mm
STYROS® 2R column.

18. 4 small molecules containing 2 polyphenyls
(Agilent sample 1080-68704) are separated on a
narrow bore column of 2.1x150 mm.
The gradient is from 60 to 100 % ACN at 40⁰ C
and monitored at 254 nm.
They include:
Application: Separation of polyphenyls on a 2.1x150 mm
STYROS® 2R column.
60 % ACN
100 % ACN
105 15 20 25 300
Product Molecular weight
Dimethyl phthalate 194.184 g/mole
Diethyl phthalate 222.24 g/mole
Biphenyl 154.21 g/mole
o-terphenyl 230.31 g/mole

19. 5 peptide standard test sample from Sigma
(H2016) were separated at acidic pH using a
simple gradient from 100 % aqueous with 0.075
% TFA to 40 % ACN, 5% H2O, 0.075 % TFA in 30
minutes at 40⁰C and monitored at 220 nm.
These peptides include:
Application: Separation of 5 peptides on a
STYROS® 2R 2.1x150 mm column at acidic pH
25105 15 200
Gly-Tyr
Val-Tyr-Val
Met-Enkephaline
Leu-Enkephaline
AngiotensinII
100% H2O, THF
40 % Acetonitrile
THF
Peptide Molecular weight
GLY-TYR MW=238.2 g/mol
VAL-TYR-VAL MW=379.5 g/mol
TYR-GLY-GLY-PHE-MET MW=573.7 g/mol as free base
TYR-GLY-GLY-PHE-LEU MW=555.60 g/mol as free base
ASP-ARG-VAL-TYR-ILE-HIS-
PRO-PHE
MW=1,046.2 g/mol as free base

20. The same peptide standard test sample from
Sigma (H2016) from the previous application were
separated at basic pH of 11.7
The gradient is the same: 100% aqueous to 40 %
ACN. However 20 mM of NH4OH was used instead
of THF. Monitored at 220 nm.
The elution is different and faster.
The separation at such high pH’s are common with
polymerics with proper composition and without
the presence of organics as there is no possibility
of phase collapse that occurs with C18 silica.
Application: Separation of 5 peptides on a
STYROS® 2R 2.1x150 mm column at basic pH
Gly-Tyr
Val-Tyr-Val
Met-Enkephaline
Leu-Enkephaline
AngiotensinII
1042 5 80 12
100% H2O,
NH4OH
40 % Acetonitrile,
NH4OH

21. Application: Separation of 5 peptides
on a STYROS® 2R 2.1x250 mm column at 80⁰C
25105 15 200 30
Gly-Tyr
Val-Tyr-Val
Met-Enkephaline
Leu-Enkephaline
AngiotensinII
100% H2O, THF
40 % Acetonitrile
THF
35 40 45 50
The previous peptide standard from Sigma
(H2016) were separated on a longer column of
250 mm at 80⁰C.
It is monitored at 220 nm.
The back pressure is now only 50 bar at the start
of the gradient with 100 % H2O.
The column does withstand the high
temperature as well as full aqueous settings
without affecting the detection.
This separation is done at acidic pH with TFA. It
can also be run at basic pH’s depending of the
targeted solutes.

22. Application: Separation of 10 peptides
on a STYROS® 2R 2.1x150 mm column 40⁰C
521 3 40 6 7 8 9 10
19 % Acetonitrile
THF
40 % Acetonitrile
THF
10 peptide standard from Agilent (5190-0583) were
separated on 2.1x150 mm STYROS® 2R column at 40⁰C
and monitored at 220 nm.
The back pressure is now only 50 bar at the start of the
gradient with 19 % ACN.
The separation can also be run at basic pH’s to alter the
retention of any targeted compound.
Peptide Molecular weight (Da)
Bradykinin frag 1-7 756.85
Bradykinin 1,060.21
Angiotensin II (human) 1,045.53
Neurotensin 1,672.92
Angiotensin I (human ) 1,296.48
Renin substrate porcine 1,759.01
[Ace-F-3,-2H-1]Angiotensinogen 2,231.61
Ser/Thr Protein Phosphatase (15-31) 1,952.39
[F14]Set/Thr Protein 2,099.00
Melittin (honey bee venom) 2,846.46

23. Separation of 9 Phenones from Agilent (part
number 5188-6529) on a STYROS® 2R narrow
bore of 2.1mm ID and 250 mm long column
were performed.
The separation was monitored at 250 nm on an
Agilent 1290 Infinity at 40 ⁰C using a gradient of
60 to 100% Acetonitrile in 30 minutes.
The pressure drop of the column is only 60 bar
at the start of the gradient.
This type of low pressure drop are the
particularity of Simulated Monolith™ columns.
Application: Separation of 9 Phenones
on a STYROS® 2R 2.1x250 mm column at 40⁰C
60%
Acetonitrile
100 % Acetonitrile
Heptanophenone
Acetophenone
Propiophenone
Butyrophenone
Valerophenone
Hexanophenone
Octanophenone
Benzophenone
Acetanilide
105 15 20 25 300 35

24. Protein standard mixture from Sigma were
separated on a STYROS® 2R 2.1x50 mm
Simulated Monolith™ column at 40⁰C and 0.2
ml/min. It was monitored at 215 nm.
Application: Separation of standard proteins from Sigma
on a STYROS® 2R 2.1x50 mm column at 40⁰C
RibonucleaseA
15%
Acetonitrile
80 % Acetonitrile
21 3 4 5 60 7 8
Cytochromec
Holo-transferrin
Apomyoglobulin
Proteins Molecular Mass
Ribonuclease A Between 13.7 and 14.7 kDa.
Cytochrome c Around 12 kDa
Holo-transferrin 76-81 kDa
Apo myoglobin 16.952 kDa
The solutes sizes have now increased without
the need of using a different ligand or a
different pore size column.

25. Using the previous conditions and increasing
the flow rate to 1 ml/min, that is an increase
from about 4,000 cm/hr. to 20,000 cm/hr. of
linear velocity, the resolution increases while the
separation time decreases at the same time.
The gradient is now shallower.
The Simulated Monolith™ STYROS® 2R
generates only 135 bar of back pressure at the
start of the gradient.
Application: Separation of the previous sample at
higher speed.
RibonucleaseA
15%
Acetonitrile
80 % Acetonitrile
21 3 4 5 60 7 8
Cytochromec
Holo-transferrin
Apomyoglobulin

26. The volumetric flow of 1 ml/min is maintained in
the previous application.
The slope of the gradient was increased to 4
minutes instead of 10.
The separation remains baseline. The time
however decreases from the original 6 minutes
to around 2.2 minutes.
This is only possible with low back pressure
columns such as Simulated Monolith™
STYROS®.
RibonucleaseA
Cytochromec
Holo-transferrin
Apomyoglobulin
1 2 30 4 4.5
15%
Acetonitrile
80 % Acetonitrile
Application: Reducing retention time by increasing
the slope of the gradient.

27. Conclusion
With the focus on the appropriate media for chromatography hyphenated
with mass spectroscopy we have suggested polymeric Simulated
Monolith™ to avoid any leaching as well as the option of using reduced
bore size columns such as narrow bore.
A number of applications were highlighted to demonstrate the universality
of the column and therefore the irrelevance of having pores of different
sizes for different molecules.
In our future presentation longer columns with smaller bore (microbore
and capillary) will be explored to address the need for detecting even
smaller and more important components of a mixture.

28. Conclusion
The end user should also feel confident that
STYROS® Simulated-Monolith™ products offer a
continuum should he needs to move to higher
scale operations and use normal bore or large
bore columns.
This chromatogram shows a normal bore column
of 4.6 mm ID using the same sample used with a
narrow bore column with the same results using
25 µl of the protein mixture instead of 2 µl with
the narrow bore column.
Lower pressure drop of the column translates into
higher number of columns for Simulated Moving
Bed operations.
0 2 min3 4 5 61
RibonucleaseA
Cytochromec
Holo-transferrin
Apomyoglobulin
15%
Acetonitrile
50 % Acetonitrile