Why polymerics as hplc media and why simulated

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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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.



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.



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.


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.



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.


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.



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.



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™.


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.


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.

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.

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.


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


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

Methyl Paraben Et

hyl Paraben

Propyl Paraben

Butyl Paraben

2 3 41 5 6

75% MeOH

100 % MeOH

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

Methyl Paraben

Ethyl Paraben

Propyl Paraben

Butyl Paraben

75% MeOH

100 % MeOH

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

Methyl Paraben

Ethyl Paraben

Propyl Paraben

Butyl Paraben

Heptyl Paraben

Phenol

Uracil

Application: Separation of 7 components on a 2.1x50 mm STYROS® 2R column.

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 weightDimethyl phthalate 194.184 g/moleDiethyl phthalate 222.24 g/moleBiphenyl 154.21 g/moleo-terphenyl 230.31 g/mole

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

Angiotensin

II

100% H2O, THF

40 % AcetonitrileTHF

Peptide Molecular weightGLY-TYR MW=238.2 g/molVAL-TYR-VAL MW=379.5 g/molTYR-GLY-GLY-PHE-MET MW=573.7 g/mol as free baseTYR-GLY-GLY-PHE-LEU MW=555.60 g/mol as free

baseASP-ARG-VAL-TYR-ILE-HIS-PRO-PHE

MW=1,046.2 g/mol as free base

The same peptide standard test sample from Sigma (H2016) from the previous application were separated at basic pH of 11.7The 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

Angiotensin

II

1042 5 80 12

100% H2O, NH4OH

40 % Acetonitrile,NH4OH

Application: Separation of 5 peptideson a STYROS® 2R 2.1x250 mm column

at 80⁰C

25105 15 200 30

Gly-Tyr

Val-Tyr-ValMet-Enkephaline

Leu-Enkephaline

Angiotensin

II

100% H2O, 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.

Application: Separation of 10 peptideson a STYROS® 2R 2.1x150 mm column

40⁰C

521 3 40 6 7 8 9 10



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

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

Benzopheno

ne

Acetanilide

105 15 20 25 300 35

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

Ribonuclease A

15% Acetonitrile

80 % Acetonitrile

21 3 4 5 60 7 8

Cytochrome c

Holo-transferrin

Apom

yoglobulin

Proteins Molecular MassRibonuclease A Between 13.7 and 14.7 kDa.Cytochrome c Around 12 kDaHolo-transferrin 76-81 kDaApo myoglobin 16.952 kDa

The solutes sizes have now increased without the need of using a different ligand or a different pore size column.

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.

Ribonuclease A

15% Acetonitrile

80 % Acetonitrile

21 3 4 5 60 7 8

Cytochrome c

Holo-transferrin

Apom

yoglobulin

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®.

Ribonuclease A

Cytochrome c

Holo-transferrin

Apom

yoglobulin

1 2 30 4 4.5

15% Acetonitrile

80 % Acetonitrile

Application: Reducing retention time by increasing the slope of the

gradient.

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.

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

Ribonuclease A

Cytochrome c

Holo-transferrin

Apom

yoglob

ulin

15% Acetonitrile

50 % Acetonitrile

Date post:	12-Apr-2017
Category:	Technology
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Why polymerics as hplc media and why simulated

Technology