Bachelor Thesis Scheikunde

Optimization and development of a second-dimension separation

method for the analysis of polymer nanoparticles by

comprehensive two-dimensional liquid chromatography

Door

Ruben Kers

1 juli 2016

Studentnummer

10499776

Onderzoeksinstituut Verantwoordelijke docent

Van ’t Hoff Institute for Molecular Sciences Prof. dr. ir. P.J. Schoenmakers

Onderzoeksgroep Begeleider

Analytical Chemistry Group B.W.J. Pirok, MSc.

2

Table of contents

List of Abbreviations .............................................................................................................................. 4

Abstract / Samenvatting .......................................................................................................................... 5

1. Introduction ......................................................................................................................................... 7

2. Theory ................................................................................................................................................. 9

2.1 Liquid chromatography (LC) ........................................................................................................ 9

2.1.1 The principle of LC ............................................................................................................... 9

2.1.2 Resolution and band broadening ......................................................................................... 10

2.2 Size-exclusion chromatography (SEC) ....................................................................................... 11

2.2.1 The principle of SEC ........................................................................................................... 11

2.2.2 Practical aspects of SEC ...................................................................................................... 12

2.2.3 Core-shell columns .............................................................................................................. 15

2.3 Hydrodynamic Chromatography (HDC) .................................................................................... 15

2.3.1 Principle of HDC ................................................................................................................. 15

2.3.2 Practical aspects of HDC ..................................................................................................... 17

3. Experimental Section ........................................................................................................................ 18

3.1 Chemicals ................................................................................................................................... 18

3.2 Instrumental ................................................................................................................................ 18

3.3 Analytical Methods ..................................................................................................................... 19

3.3.1 Sample Preparation ............................................................................................................. 19

3.3.2 Separation of PLGA with different SEC columns .............................................................. 21

3.3.3 Calibration of size-exclusion columns ................................................................................ 21

3.3.4 Analysis of large polydispersity PS ..................................................................................... 22

3.3.4 HDC column testing ............................................................................................................ 22

4. Results & Discussion ........................................................................................................................ 23

4.1 Separation of PLGA with PLGel columns ................................................................................. 23

4.1.1 Column efficiency determination with PLGA .................................................................... 23

4.1.2 SEC calibration with polystyrene and polymethylmethacrylate ......................................... 28

4.2 Separation of PLGA mixture with experimental core-shell columns ......................................... 32

4.2.1 Column efficiency determination with PLGA .................................................................... 32

4.2.2 SEC calibration with polymethylmethacrylate .................................................................... 34

4.2.3 SEC Calibration with 3 coupled columns ........................................................................... 35

4.2.4 Analysis of large polydispersity PS ..................................................................................... 36

4.2.5 Concluding remarks regarding SEC analysis ...................................................................... 37

4.3 HDC Measurements ................................................................................................................... 38

5. Conclusion ........................................................................................................................................ 41

6. Future Prospects ................................................................................................................................ 42

References ............................................................................................................................................. 43

3

Acknowledgement

First of all, I would like to thank Bob Pirok, MSc. for his guidance and supervision during my

bachelor project, his insight and willingness to share his views with me on the different

problems were most supportive and certainly increased the quality of my work. Secondly, I

want to thank Fleur van Beek, MSc. for being accessible and helpful with my project whenever

I was in need of it. I would also like to thank prof. dr. ir. Peter Schoenmakers and dr. Wim Kok

for allowing me to do my project at the Analytical Chemistry Group at the UvA. Finally, I

would like to thank Serafine, Pascal, Charlotte and Leon for helping me with the practical

aspects of HPLC.

4

List of Abbreviations

3-NBS acid 3-Nitrobenzenesulfonic acid

BHT Butylated hydroxytoluene

Dc Column diameter

DAD Diode array detector

e Porosity

γ Unadjusted relative retention

GFC Gel filtration chromatography

GPC Gel permeation chromatography

H Plate height

HDC Hydrodynamic chromatography

HPLC High-performance liquid chromatography

IEC Ion-exchange chromatography

L Column length

LC Liquid chromatography

LC×LC Two-dimensional liquid chromatography

N Plate number

NPLC Normal-phase liquid chromatography

PLGA Poly(lactic-co-glycolic acid)

PMMA Polymethylmethacrylate

PS Polystyrene

RID Refractive-index detector

RPLC Reversed-phase liquid chromatography

SC Slalom chromatography

SEC Size-exclusion chromatography

tR Retention time

THF Tetrahydrofuran

Ux Linear flowrate

UHPLC Ultra-high performance liquid chromatography

V0 Void volume

VR Retention volume

5

Abstract

The separation of molecules by size-exclusion chromatography (SEC) is nowadays a widely

established technique in the determination of molecular weight distribution of polymers, but

combining SEC with hydrodynamic chromatography (HDC) in one comprehensive analytical

two-dimensional LC system (LC×LC) for the determination of polymer nanoparticles has not

yet been achieved. In this study, which is part of the MAnIAC project, research is conducted

towards the development of a second dimension method for this comprehensive analytical

system, which is based on SEC and where HDC is applied to verify the proposed method. From

this study it can be concluded that columns with high plate numbers have to be used in order

to achieve good resolution when separating low-molecular weight polymers. It has been found

that using multiple columns and certain core-shell columns result in sufficient separation

efficiency to give good resolution between the investigated samples. HDC analysis on

polymers has not been performed as method verification due to insufficient column

performance.

Samenvatting

Vloeistofchromatografie is een scheidingsmethode die binnen de analytische chemie veel

wordt toegepast voor de analyse van chemische stofmengsels. Bij vloeistofchromatografie

wordt een scheiding uitgevoerd in een kolom, gevuld met deeltjes, waar doorheen een continue

stroom van vloeistof loopt. Daarin wordt het te analyseren monster opgelost. Deze oplossing

komt uiteindelijk in een detector terecht, waarbij - in het ideale geval - de individuele stoffen

kunnen worden onderscheiden. Een scheidingskolom bestaat meestal uit deeltjes van vaste stof,

zoals silica of koolstof. Bepaalde stoffen hebben een hogere affiniteit of passen beter door de

poriën van de deeltjes van de kolom, waardoor er een scheiding wordt gedaan op basis van

bijvoorbeeld hydrofobiteit, lading of grootte van moleculen.

In de analyse en scheiding van polymeren voor de bepaling van hun molmassa, wordt

tegenwoordig vaak gebruik gemaakt van methoden als size exclusionchromatografie (SEC) en

hydrodynamische chromatografie (HDC). Het is echter nog moeilijk om beide

scheidingsmethoden in één systeem aan elkaar te koppelen. In theorie zou daardoor meer

informatie verkregen kunnen worden over de te analyseren stof dan wanneer slechts één

methode wordt toegepast. In het MAnIAC project wordt getracht een systeem te ontwerpen dat

dit soort analyses uit kan voeren en wel met behulp van HDC en SEC.

6

In dit bachelor project ligt de focus op het vinden van een goede chromatografische methode

voor het scheiden van polymeren met behulp van SEC. Hierbij wordt vooral gekeken naar welk

type kolommen het beste werken voor het scheiden van polymeren met verschillende

deeltjesgrootte. Het is gebleken dat een goede scheiding plaatsvindt tussen polymeren met

relatief lage molmassa wanneer meerdere scheidingskolommen worden toegepast of

kolommen worden gebruikt die deeltjes bevatten met een harde kern en poreuze laag. Ook is

geprobeerd om HDC toe te passen voor de verificatie van de SEC-methode. Uit de resultaten

is gebleken dat de HDC-kolom in onvoldoende mate functioneerde om goede analyse uit te

voeren. Voor vervolgonderzoek zou dus de methode geverifieerd kunnen worden met HDC als

scheidingsmethode.

Chapter 1: Introduction

7

1. Introduction

Polymers are large molecules that play key roles in everyday life and are interesting due to

their wide range of properties. Polymers consist of an array of repeating subunits of carbon

backbone which can contain various functional groups and heteroatoms. Examples of widely

known polymers are DNA, as a naturally occurring polymer and polystyrene as a synthetically

occurring polymer. Polymers are used in a broad variety of applications due to their variable

chain size, hydrophobicity and functions, and are therefore incorporated in a large array of

different materials.1

An important application of polymers is the synthesis of nanoparticles, which are

particles with at least one dimension between 1 nm and 100 nm in size.2 An example of an

application of nanoparticles is in the use of paint as coatings that have anti-reflective

properties.3 These coatings are furthermore applied in for instance anti-corrosion solutions,

enhanced surface appearance for furniture and UV-protection.4 For companies that produce

coatings and the polymer nanoparticles they consist of, it is important to know the composition

of their product and its characteristics. The knowledge on how their product behaves can be

used to provide an opportunity for further development of the product. Conventionally used

analytical techniques are applied to analyze these nanoparticles and polymers, such as

chromatography.5

In the MAnIAC project, research is conducted towards the development of a

comprehensive analytical system to determine the most important characteristics of these

nanoparticles. The proposed system is portrayed in Figure 1, and is based on the principle of

two-dimensional liquid chromatography (LC×LC) where two distinct separations are

combined in one system for improved chromatographic results.

Figure 1: Proposed comprehensive analytical system under development in the MAnIAC

project.

Chapter 1: Introduction

8

In the first dimension it is envisaged to separate the polymeric nanoparticles based on their size

by using hydrodynamic chromatography (HDC). The nanoparticles are then to be dissolved in

the modulator and the products of the dissolution are analyzed by a second dimension

separation such as size-exclusion chromatography (SEC), reversed-phase liquid

chromatography (RPLC) or ion-exchange chromatography (IEC).

HDC and SEC/RPLC are the methods of choice in this comprehensive system due to

the important information that is acquired with these techniques. HDC is capable of separating

aggregated nanoparticles in aqueous solution, while SEC is highly efficient in determining the

polymers in organic solution that make up the larger nanoparticles. The proposed MAnIAC

system could therefore achieve a higher selectivity compared to currently used methods, and is

thus exceptionally interesting for certain applications in industry such as polymer

characterization.

In this study, a possible second dimension separation mechanism is investigated on

latex nanoparticles and polymers, using SEC and HDC. The results from this study can be used

to fine-tune the system operating conditions like used stationary phases, flow rates, injection

volumes, and dilution factors for the second dimension separation. In this research, the

measurements for chromatographic optimization will be conducted using polystyrene (PS),

polymethylmethacrylate (PMMA) and poly(lactic-co-glycolic acid) (PLGA) samples with

differing chain lengths to measure the response of the analytical system.

Nanoparticles will be prepared with building blocks of PLGA, by making different

known ratios of building blocks (consisting of PLGA and polyethylene glycol (PEG)) and

separating them in the first dimension of the comprehensive system with HDC in aqueous

mobile phase. The nanoparticles are then to be dissolved and separated in the second dimension

with SEC in THF mobile phase, where the original building blocks can be distinguished and

by which the method is verified.

It is expected that the separation of these polymers is challenging due to the wide

molecular weight distribution of the analyzed molecules, resulting in broad peaks and

consequently loss of resolution.

Chapter 2: Theory

9

2. Theory

2.1 Liquid chromatography (LC)

2.1.1 The principle of LC

Liquid chromatography (LC) is a widely used separation technique which separates analytes

based on their characteristics, such as hydrophobicity or molecular size. A typical LC setup

consists of a pump, column and detector. In LC, the separation occurs in the column (the

stationary phase) which consists of either a nonpolar material like C18, or a polar material like

silica for respectively reversed-phase liquid chromatography (RPLC) or normal-phase liquid

chromatography. Another mode of separation in LC is based on the molecular size of the

analytes, which is the separation mechanism that occurs in size-exclusion chromatography

(SEC) and hydrodynamic chromatography (HDC). The analyte is dissolved in the mobile

phase, which usually consists of either acetonitrile, methanol, water, THF or mixtures of

solvent that flows continuously through the system.6 In Figure 2 a typical setup of an LC

system is shown.

Figure 2: Schematic of an LC setup.7

When pressures of up to 400 bar are applied for separation in LC, the technique is called high-

performance liquid chromatography (HPLC). When operating under ultra-high pressures

(~1000 bar) the technique is referred to as ultra-high performance liquid chromatography

(UHPLC). The benefit of using high pressure is the fact that separation can be performed faster,

and smaller particles in the stationary phase can be used which increases resolution at the

expense of increased backpressure.8

Chapter 2: Theory

10

LC is nowadays used as a method of analysis or as a purification method, due to the

fractionation that occurs in the column.6 The LC output can be subsequently coupled to

different types of detection methods, like UV detection, refractive-index detection,

fluorescence detection, evaporate light scattering detection or mass spectrometry (optical

rotation detection, tandem mass spectrometry).6 In this study, HPLC is applied with UV and

refractive-index detection.

2.1.2 Resolution and band broadening

The resolution of separation in LC depends on two factors: the difference in elution time of

compounds and the band broadening of peaks. There are multiple approaches to influencing

the resolution, for example by changing the elution strength of the mobile phase, changing the

temperature of the column, changing particle size of the column and changing the column

length.6 In general, the higher the amount of theoretical plates, the better the resolution (see Eq.

(1)):

𝑅𝑒𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 √𝑁

𝛾 − (1)

Where N is the number of theoretical plates, and γ is the unadjusted relative retention.6

The amount of plates on a column is related to the length of the column and the plate height by

Eq. (2):

𝑁𝐿

𝐻 (2)

Where N is the number of plates, L is the column length and H is the plate height.6

The influence of the flow rate on the plate number and plate height in a certain column can

furthermore be described by the Van Deemter Equation (Eq. (3)):

𝐻 ≈ 𝐴 𝐵

𝑈𝑥 𝐶𝑈𝑥 (3)

Where H is the plate height, A, B and C are constants dependent on the multiple flow paths

within the column, longitudinal diffusion and equilibration time, respectively. Ux is the linear

velocity.6

Chapter 2: Theory

11

The Van Deemter Curve describes the column efficiency of the stationary phase and is

dependent on the band broadening processes that occur during separation. Band broadening

occurs for instance due to Eddy diffusion, which is related to the multiple flow paths that

molecules take when going through the column. Flowrates are different in narrow and broad

flow paths, which causes different velocities in the same column and results in band

broadening. The A-term in the Van Deemter curve describes this Eddy diffusion. Furthermore,

longitudinal diffusion causes band broadening due to dispersion of analyte within the column,

which is described by the B-term in the Van Deemter Curve. Also the resistance of the analyte

to mobile-phase mass transfer is an important cause of band broadening, which means that

solute near the particles of the packing move more slowly through the column than the solute

that is further away from the packing. This is described by the C-term of the Van Deemter

curve. The characteristics of the column packing, like particle size and pore sizes have

significant influence on band broadening processes. To optimize resolution, it is therefore

important to take into account the type of stationary phase used.9

The resolution of separations, plate number and plate height are important factors in the

development of useful LC×LC analysis methods. However, the magnitude of these effects are

often times best to be determined experimentally and therefore the aim of this study is to find

optimal conditions in which good polymer separations can be performed.

2.2 Size-exclusion chromatography (SEC)

2.2.1 The principle of SEC

Size exclusion chromatography (SEC) is a variant of chromatography in which compounds are

separated based on their size. The stationary phase consists of porous packing material, in

which small particles diffuse through pores of the column packing, whereas larger molecules

are excluded of the pores. There is no interaction with the stationary phase other than the

selective diffusion through the pores that is caused by entropic effects.9 The result is a

separation based on the difference in rate at which molecules migrate through pores of the

stationary phase particles, which depends on the molecular weight and hydrodynamic size of

the molecules.5, 10 This process of size exclusion during a SEC separation is illustrated in Figure

3.

Chapter 2: Theory

12

Figure 3: Illustrative schematic of exclusion of compounds within pores in SEC.

SEC is often applied for the determination of the average molecular weight of biopolymers and

synthetic polymers. There are various types of SEC columns, they differ in for example pore

size, column length and column diameter. In gel permeation chromatography (GPC), a

stationary phase consists of a hydrophobic gel in combination with an apolar mobile phase,

while in gel filtration chromatography (GFC), a hydrophilic packing material is used in

combination with a polar mobile phase. Packing material with distinct pore sizes are produced

for optimal separation of different polymer size ranges and there are multiple types of columns

available for a broad series of polymers.11

2.2.2 Practical aspects of SEC

A typical size determination measurement in SEC starts with the setup of a calibration curve

with known molecular weight standards. The logarithm of the molar weight is plotted against

the retention volume (VR), from which the molecular weight of the unknown sample is

calculated. A calibration curve is different for every system, due to the multiple factors

involved that determine retention in a column. Parameters which are of significant influence

on the measurements are for example the size of the packing material, the material of which

the packing material consists, the inner diameter and the length of the used columns.9

Furthermore, the hydrodynamic size of the analyzed polymers has to be taken into account and

the right polymer standards need to be used, as polymers have different ways in which they are

folded.5 Calibration measurements are applied in this study for the calculation of polymer

molar mass, and to confirm that the utilized SEC method provides good results with known

PLGA samples. An example of a SEC calibration curve is portrayed in Figure 4.

Chapter 2: Theory

13

Figure 4: Example of a SEC calibration curve of polystyrene.12

The maximum size of molecules that can be separated on a SEC column elute on the left side

of the calibration curve that is following an increasing vertical trend, which is termed the

exclusion limit of the column and is caused by the fact that those molecules are of sizes too

large to enter the pores of the stationary phase and thus go through the column without being

retained. The other end of the calibration curve is called the permeation limit, which is achieved

when molecules have such a low molar mass that they penetrate the pores of the stationary

phase completely. The t0 marker, which in this study is toluene in SEC, elutes at the permeation

limit.11 The V0 and therefore t0 in SEC can be predicted theoretically with Eq. (4):

𝑉 𝐷𝑐𝐿𝑒

𝜋 (4)

Where V0 is the void volume, dc is the column diameter, L is the length of the column and e is

the porosity.13

When molecules reach a certain hydrodynamic size above the exclusion limit of a

column, they can start to unfold and stretch within the stationary phase, which causes a

phenomenon that is called slalom chromatography (SC). SC is caused by the stretching of

molecules turning around the stationary phase packing material, which results in increased

retention and is an entirely different retention mechanism than that of SEC. The tendency in

which SC occurs in the stationary phase is dependent on the size of the analyzed molecules,

the column packing, the flow rate, solvent viscosity and temperature of the column.9 In this

study, SC is observed in the calibration of columns with polymer standards, but not during

PLGA analysis due to the small molecular weight of the samples used in this study.

Chapter 2: Theory

14

Current SEC methods show promising results in determining the characteristics of polymers,

and it is therefore an increasingly important technique in the determination of molar weight of

particles in the 100 Å to 10.000 Å linear dimension range.10 Furthermore, one of the features

of polymer analysis with SEC is that the molecular weight distribution can be determined, due

to the differing polydispersity of the polymers and elution with a Gaussian distribution. Both

biopolymers and synthetic polymers diverge from narrowly to broadly polydisperse, which

results in varying broadness of peaks on the chromatogram. Due to the wide distribution of the

molar weight, there is potential loss of resolution which poses a challenge in choosing the right

stationary phases in SEC.9 Figure 5 depicts a mass spectrum of polystyrene, showing the

distribution of polymer molecular weight caused by the polydispersity, resulting in broad peaks

in SEC chromatograms.

Figure 5: MALDI-ToF-MS spectrum of polystyrene with a molecular weight of 5050 g/mol and

polydispersity of 1.05.14

Chapter 2: Theory

15

2.2.3 Core-shell columns

An emerging new technology in HPLC is the application of core-shell particles in SEC

separations. Core-shell particles are particles of porous silica or C18 material which is wrapped

around a solid core, resulting in a particle size of ~2.6 μm, but which has the efficiency of a

sub-2 μm particle size column. This increased efficiency is induced by the lower pore volume

in core-shell columns, which lowers the longitudinal diffusion in the column, but also decreases

the A- and the C-term in the Van Deemter curve due to faster mass transfer and rougher surface

of the particles. The disadvantage of core-shell columns is the fact that the pore volume is

significantly smaller than for columns with completely porous particles, which retarded the use

of core-shell columns due to fear of lower separation capability. But on the contrary, the latest

studies on core-shell columns show that these stationary phases are readily capable of excellent

resolution and fast separations on UHPLC devices.

In this study, experimental Phenomenex silica and C18 core-shell columns will be

applied in the separation of polymers, to investigate if these core-shell columns are indeed

capable of increased resolution in PLGA separations.

2.3 Hydrodynamic Chromatography (HDC)

2.3.1 Principle of HDC

A chromatographic method which is exceptionally useful in the determination of nanosize

particles is HDC, in which non-porous inert particles in packed columns or open tube capillary

columns are applied to induce separation, and in which the size, the shape and the structure of

analytes can be determined, when coupled to a variety of detection methods. The separation in

HDC is based on the Poiseuille-like (parabolic) flow profile, which is the result of different

flow lines within the column. Flow lines that are closer to the walls of the column or to the

stationary phase particles move slower than flow lines in the middle of the column. Due to the

capability of smaller molecules to approach the walls or particles of the column more closely,

they elute at a later time compared to the larger particles that experience a greater net flow.10

Figure 6 on the next page depicts the mechanism of HDC separation.

Chapter 2: Theory

16

Figure 6: Mechanism of HDC separation in an open tubular column and a packed column.10

HDC is originally an early established technique in chromatography, but has undergone a

resurgence because of its capability in separating molecules with higher molar mass than

SEC.10 The use of HDC columns is attractive when large hydrophobic polymers are being

analyzed in aqueous samples, due to micelle formation which is the result of the aggregation

of hydrophobic material in order to reduce its contact area with the hydrophilic environment.

The micelles itself are larger particles than the un-aggregated polymers, scaling up their

hydrodynamic size into the nanoparticle range and thus making HDC an excellent technique

for separation.10 Figure 7 portrays a schematic of a polymer micelle.

Figure 7: Micelle which is build up from polymers in aqueous solution.16

Chapter 2: Theory

17

2.3.2 Practical aspects of HDC

In HDC, packed columns are more conventionally used due to the narrow channel that is

necessary in open HDC columns. Using open HDC columns is difficult, which is especially

caused by the requirement of low injection volumes (~1 µL) to avoid column overloading.5

HDC effects arise during separations of very large molecules and for columns with

small pore sizes, which is a widely occurring phenomenon due to the fact that at a certain point

of molecular weight the molecules approach the exclusion limit and will stop transfering

through the porous stationary phase. It is therefore possible to perform HDC with size-

exclusion columns. The rate at which HDC effects occur depends on the ratio of the pore size

of the particles to the diameter of the particles (Rp/dp), a decrease in this ratio results in less

HDC effects in the stationary phase and a more dominant SEC separation mechanism during

the separation.9 For instance in this study, HDC effects are induced with experimental SEC

columns that contain small porous particles, on molecules with high molecular weight.

In the determination of molar weight distribution of polymers with HDC, it is standard

procedure to first construct a calibration. A calibration is performed by injecting polymers of

known sizes and measuring the retention time of the polymer with corresponding molar weight,

just like in SEC. An illustration of different calibration curve regions for HDC, SEC and SC is

given below in Figure 8.17

Figure 8: Calibration with polystyrene showing the specific molar weight regions in which

they are separated using SEC, HDC and SC mechanisms.17

Chapter 3: Experimental Section

18

3. Experimental Section

3.1 Chemicals

Tetrahydrofuran (THF, AR-grade, stabilized and HPLC-S grade, unstabilized) was obtained

from Biosolve B.V. (Valkenswaard, the Netherlands) and used for the preparation of samples

and as SEC mobile phase. Butylated hydroxytoluene (BHT) was obtained from Sigma Aldrich

(St. Louis, MO, USA) and was applied as HPLC-S grade THF stabilizer. Toluene was acquired

from Biosolve B.V. (Valkenswaard, the Netherlands) and was applied as SEC marker.

Deionised water (Arium 611UV, Sartorius, R=18.2 MΩ cm-1, Germany) was prepared as HDC

mobile phase and applied in sample dilutions. HDC buffer was obtained from Agilent

Technologies (Amstelveen, the Netherlands) and used as HDC mobile phase surfactant.

Potassium dichromate was purchased from Sigma Aldrich (St. Louis, MO, USA), uracil was

obtained from Sigma Aldrich (St. Louis, MO, USA) and 3-nitrobenzenesulfonic acid (3-NBS

acid) was received from Sigma Aldrich (St. Louis, MO, USA), which were used as an HDC

marker. Polystyrene (PS) and poly(methylmethacrylate) (PMMA) samples with different

polymer length were obtained from Polymer Laboratories (Amherst, U.S.). Poly(lactic-co-

glycolic acid) (PLGA) samples with a molar weight distribution of 21 kDa and 10.5 kDa and

large polydispersity PS with molecular weight distribution of 200 kDa were received from

DSM Coating Resins (Waalwijk, the Netherlands).

3.2 Instrumental

All HPLC measurements using RID detection were conducted on a combined Agilent 1100

series and 1260 series LC setup, which consisted of an 1100 series nanopump (G2226A), an

1100 series refractive-index detector (G1362A), an Infinity 1260 series degasser (G1322A) and

an 1100 series thermostatted column compartment (G1316A). Manual injection was used with

a 20 μL sample loop installed.

All HPLC measurements using UV detection were conducted on a combined Agilent

1100 series, 1260 series and 1290 series LC setup, which consisted of a 1100 series quaternary

pump (G1311A), a 1290 series diode array detector (G4212A), an Infinity 1260 series degasser

(G1322A), and an 1100 series thermostatted column compartment (G1316A). An autosampler

was used with 20 µL injections, consisting of an 1100 series ALS (G1313A).

Chapter 3: Experimental Section

19

All UHPLC measurements were conducted on an Agilent 1290 Infinity LC system, which

consisted of an Infinity 1290 binary pump (G4220A), an Infinity 1290 diode-array detector

(G4212A), an Infinity 1290 autosampler and an Infinity 1290 thermostatted column

compartment (G1316C).

3.3 Analytical Methods

3.3.1 Sample Preparation

SEC calibration standards were prepared separately by dissolving 100 mg of either PS or

PMMA with known molecular weight in 5 mL of THF, to a concentration of 20 mg/mL. The

SEC calibration stock solutions were subsequently diluted in THF to a concentration of 0.5

mg/mL. The molecular weight of both used PS and PMMA standards are given on the next

page in Table 1.

Chapter 3: Experimental Section

20

Table 1: Molecular weight of the used PS and PMMA SEC calibration standards.

PS

Standard

Molar

Weight

(g/mol)

PMMA

Standard

Molar Weight

(g/mol)

PS01 580 PMMA01 620

PS02 980 PMMA02 1140

PS03 1990 PMMA03 1310

PS09 9920 PMMA04 1960

PS10 13880 PMMA05 2000

PS11 19880 PMMA06 2870

PS12 52400 PMMA07 6820

PS13 70950 PMMA08 10260

PS16 299400 PMMA09 14920

PS19 735500 PMMA10 24830

PS21 1373000 PMMA11 30690

PS22 2061000 PMMA12 49600

PS23 2536000 PMMA13 79500

PS24 3053000 PMMA14 100000

PS25 3743000 PMMA15 141500

PS26 7450000 PMMA16 211400

PS27 13000000 PMMA17 300300

- - PMMA18 518900

- - PMMA19 659000

- - PMMA20 948500

- - PMMA21 1250000

PLGA samples for SEC were prepared by dissolving 64.3 mg of (PLGA(7.5 kDa))2-PEG(6

kDa) (PLGA 21 kDa) in 1 mL THF, 63.4 mg (PLGA(3.75kDa))2-PEG(3 kDa) (PLGA 10.5

kDa) in 1 mL THF and dissolving 30.2 mg PLGA (21 kDa) combined with 31.1 mg PLGA

(10.5 kDa) in 1 mL THF. The samples were subsequently diluted 10, 50 and 100 times in THF

to a volume of 1 mL.

Chapter 3: Experimental Section

21

A vial of 3-Nitrobenzenesulfonic acid (3-NBS acid) was prepared to a concentration of 2.0

mg/mL in HDC buffer. 301 mg Potassium dichromate was dissolved in 5 mL HDC buffer

after which the solution was diluted to a concentration of 0.301 mg/mL in HDC buffer. 200

mg Uracil was dissolved in 5 mL warm HDC buffer and diluted to a concentration of 2.0

mg/mL. The prepared solutions were used as HDC t0 marker.

3.3.2 Separation of PLGA with different SEC columns

To determine whether the PLGA particles could efficiently be separated with the available

PLGel columns, different PLGA sample mixtures were injected into the HPLC system using

PLGel Mixed-E (7.5x100 mm, 3 μm), PLGel Mixed-D (7.5x300 mm, 5 μm) and PLGel Mixed

(7.5x600 mm) columns, using refractive-index detection. The flowrate was set to 1.0 mL/min.

The mobile phase consisted of 100% THF (AR-grade, stabilized). The column compartment

and the RID had a temperature set to 30 °C. The injection volume was 20 μL for every

measurement.

To test the efficiency of a Phenomenex core-shell B109 column (4.6x150 mm, 2.6 μm,

XB-C18), the different PLGA sample mixtures were injected into the HPLC system using

refractive-index detection. The flowrate was set to 0.2 mL/min. The mobile phase consisted of

100% THF (HPLC-S grade, stabilized). The column compartment and the RID had a

temperature set to 30 °C. The injection volume was 20 µL for every measurement.

3.3.3 Calibration of size-exclusion columns

Calibrations with PS and PMMA were performed by injecting every PS and PMMA standard

(see Table 1) into the HPLC setup with RID detection. A PMMA and PS calibration was

conducted with PLGel Mixed (7.5x600 mm), with a flowrate of 1.0 mL/min for every

measurement. The mobile phase consisted of 100% THF (AR-grade, Stabilized). The used

column and RID temperature was set to 30 °C. The injection volume was 20 µL for every

measurement.

A calibration was performed on a Phenomenex core-shell B109 column (4.6x150 mm,

2.6 μm, XB-C18), by injecting PMMA standards (see Table 1) with the HPLC setup on RID

detection. A flowrate of 0.2 mL/min was used for every measurement. The mobile phase

consisted of 100% THF (HPLC-S grade, unstabilized) with added BHT (to 200 ppm). The used

column and RID temperature was set to 30 °C. The injection volume was 20 µL for every

measurement.

Chapter 3: Experimental Section

22

A calibration with PS (see Table 1) was performed on three coupled Phenomenex core-shell

B111 (4.6x150 mm, 3.6 μm, XB-C18), B104 (4.6x150 mm, 2.6 μm, silica) and B109 (4.6x150

mm, 2.6 μm, XB-C18) columns, on an UHPLC system with UV detection. The flowrate was set

to 2.00 mL/min for every measurement. The mobile phase consisted of 100% THF (HPLC-S

grade, unstabilized) with added BHT (to 200 ppm). The column compartment had a

temperature set to 25 °C. The injection volume was 1 µL for every measurement.

3.3.4 Analysis of large polydispersity PS

To see how the Phenomenex core-shell B111, B104 and B109 columns respond to large

polydispersity polymers, a large polydispersity PS sample with a molecular weight of 200.000

g/mol was injected on an UHPLC system with UV detection. The flowrate was set to 2.00

mL/min for every measurement. The mobile phase consisted of 100% THF (HPLC-S grade,

unstabilized) with added BHT (to 200 ppm). The column compartment had a temperature set

to 25 °C. The injection volume was 1 µL for every measurement.

3.3.4 HDC column testing

An injection with 3-NBS acid and uracil was performed using an Agilent PL-PSDA Cartridge

Type 1 HDC column (7.5x800 mm), on an HPLC system with UV detection. The flowrate

was set to 1.6 mL/min for the 3-NBS acid injection, uracil injection and a blank sample,

containing acidified HDC buffer (pH ~3.2). The mobile phase consisted of acidified HDC

buffer. The used column temperature was set to 25 °C. The injection volume was 0.5 μL for

3-NBS acid and 5 μL for uracil.

An injection with potassium dichromate was performed using an Agilent PL-PSDA

Cartridge Type 1 HDC column (7.5x800 mm), on an HPLC system with UV detection. The

flowrate was set to 1.7 mL/min for both the potassium dichromate injection and the blank

sample, containing HDC buffer (pH ~7). The mobile phase consisted of HDC buffer. The

used column temperature was set to 25 °C. The injection volume was 10 μL.

Chapter 4: Results & Discussion

23

4. Results & Discussion

4.1 Separation of PLGA with PLGel columns

4.1.1 Column efficiency determination with PLGA

Figure 9: Chromatogram of PLGA mixture, blank THF sample and toluene marker, separated

with a PLGel Mixed-E column.

Figure 9 shows the chromatogram of the PLGA mixture sample separated on a PLGel Mixed-

E column. As illustrated in the figure, there is a signal visible at 1.99 min. which is assigned to

the PLGA sample, it is absent in the blank sample and elutes before the toluene marker, which

elutes at 3.09 min. The t0 marker has a retention time that sufficiently corresponds to the

theoretically predicted value of 3.27 min., which is the retention time at the permeation limit

and is calculated according to Eq. (4). The positive signal at 2.73 min. and the strong negative

signals are suspected system peaks. The positive system peaks are most likely the result of

components present in the sample but absent in the eluent, which could be due to either

contamination of the sample or the formation of peroxides in the sample that form when the

THF reacts with oxygen. The negative signals are caused by compounds in the eluent that are

not present in the injection, which could for example be peroxides that exist in a different

concentration than in the sample.18 Figure 10 depicts the chromatogram of this PLGA sample.

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0 1 2 3 4 5 6

Sign

al (

nR

IU)

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PLGel Mixed-E Column

Blank

t0

PLGA

Chapter 4: Results & Discussion

24

Figure 10: Chromatogram of PLGA mixture, separated with a PLGel Mixed-E column.

There is no separation between both PLGA samples visible from this chromatogram, but a

broad distribution is observed which is assumed to correspond to both PLGA samples. From

this result it can be concluded that the used column was not capable of giving sufficient

resolution for the separation between the PLGA samples labeled to be 10.5 kDa and 21 kDa.

To improve separation using this type of column, a column with a larger length could

potentially be applied for the separation of the PLGA particles, to increase the plate number

according to Eq. (2). To confirm that the low resolution is the cause of low column efficiency

in the separation of these PLGA samples, a calibration curve could be constructed, which shows

that an insufficient resolution is achieved. This particular calibration curve of the PLGel Mixed-

E column was not made in this project.

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200

400

600

800

1,5 1,6 1,7 1,8 1,9 2 2,1 2,2 2,3 2,4 2,5

Sign

al (

nR

IU)

Time (min)

PLGel Mixed-E Column

PLGA

Chapter 4: Results & Discussion

25

Figure 11: Chromatogram of PLGA mixture, blank THF sample and toluene marker,

separated with a PLGel Mixed-D column.

Figure 11 shows the chromatogram of the same PLGA sample mixture, this time separated on

a PLGel Mixed-D column, which was tested due to its molar weight operating range of 200-

400.000 g/mol and longer column length compared to the PLGel Mixed-E column, which in

theory results in a higher plate number.19 It is assumed that the signal at 6.76 min. is

corresponding to the PLGA mixture, due to its absence in the blank and because of elution

before the toluene marker at 9.47 min. As can be observed from this figure, the PLGA sample

mixture once again shows a broad distribution which is expected due to the polydispersity of

PLGA polymers. Furthermore, it is assumed that the negative signals shown in the

chromatogram are system peaks which is most likely the result of compounds in the THF

mobile phase, which might be due to the formation of peroxides. Furthermore, from this figure

it can be seen that the toluene marker has a significantly higher intensity of negative signals

compared to the blank and PLGA samples. This can be related to the fact that the used vial

with toluene, was prepared from a different bottle of THF solvent than the blank and PLGA

sample. From this chromatogram it can be concluded that the composition of this THF was

different. In Figure 12 below, a chromatogram is given which highlights the PLGA signal.

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3000

4000

0 2 4 6 8 10 12 14 16

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Time (min)

PLGel Mixed-D Column

Blank

t0

PLGA

Chapter 4: Results & Discussion

26

Figure 12: Chromatogram of PLGA mixture, separated with a PLGel Mixed-D column.

Using this chromatogram, it can be concluded that the PLGA mixture cannot be sufficiently

separated with this column, due to the substantial broadness of the signals, resulting in low

resolution. There is a small bump visible at 6.5 min which might correspond to the larger

molecular weight PLGA sample, but there is a substantial overlap between both compounds.

To increase separation, a column with higher column efficiency for this molecular weight range

should be used.

Figure 13: Chromatogram of PLGA mixture, blank THF sample and toluene marker,

separated with a PLGel Mixed double column.

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50

100

150

200

250

300

5 5,5 6 6,5 7 7,5 8

Inte

nsi

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PLGel Mixed-D Column

PLGA

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2000

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10000

0 5 10 15 20 25

Inte

nsi

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Time (min)

PLGel Mixed 2 columns

Blank

t0

PLGA

Chapter 4: Results & Discussion

27

Figure 13 shows the chromatogram of the PLGA mixture, separated on two PLGel mixed

columns. As can be observed from the chromatogram, the signal at 14.5 min corresponds to

the PLGA mixture, which is determined from the PLGA sample and the blank mixture. It is

assumed that the positive signal at 18.8 min and the negative signals are system peaks, which

might be the result of the formation of peroxides due to aging of the THF solvent. Figure 14

below highlights the PLGA signal.

Figure 14: Chromatogram of the PLGA mixture and individual injections, separated with a

PLGel Mixed Double column.

As illustrated in this chromatogram, there is a broad signal visible which is assigned to the 21

kDa PLGA sample, and a signal with less intensity at ~15.5 min. that overlaps with this broad

peak of higher intensity. It is assumed this signal corresponds to the PLGA with a mass of 10.5

kDa, due to the overlap with the individual injection. The lower intensity of this smaller PLGA

sample might be the result of either higher polydispersity, pipetting error or insufficient

homogenization of both samples. Nevertheless, these results suggest that the PLGel Mixed

double column can induce adequate separation of these PLGA particles.

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300

400

12 13 14 15 16 17

Inte

nsi

ty (

nR

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Time (min)

PLGel Mixed 2 columns

PLGA 10.5 kDa

PLGA 21 kDa

PLGA Mixture

Chapter 4: Results & Discussion

28

4.1.2 SEC calibration with polystyrene and polymethylmethacrylate

Figure 15: Chromatograms of PMMA with molar weight distributions of 2000 and 24830

g/mol.

Figure 15 depicts the chromatogram of PMMA polymer calibration standards with molar

weight distributions of 2000 g/mol, and 24830 g/mol used with a double column of PLGel

Mixed (7.5x600 mm), recorded with RID detection. As portrayed in the chromatogram, there

are signals at 14.3 and 16.2 min. which have been assigned to the PMMA standards. The

positive signal at 18.0 min. and the negative signals are assumed to be system peaks related to

degradation of THF to peroxides in the prepared samples and the mobile phase, and correspond

to the blank sample which consisted of THF. Furthermore, as can be seen from this

chromatogram, the signal corresponding to the smaller polymer standard elutes after the larger

polymer standard, which is expected due to the fact that smaller molecules enter the pores of

the packing more easily and are therefore more retained in SEC.

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0 5 10 15 20 25

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PLGel Mixed 2 Columns

PMMA 2000 g/mol

PMMA 24830 g/mol

Chapter 4: Results & Discussion

29

Figure 16: Chromatograms of PS with molar weight distributions of 1990 and 19880 g/mol.

Figure 16 depicts the chromatogram of PS calibration standards with molar weight

distributions of 1990 and 19880 g/mol, recorded with the same setup as the PMMA calibration.

As is depicted in this figure, there are positive signals at 13.8 and 16.0 min. corresponding to

the PS samples, which is determined from the subtraction of the blank sample. The positive

signal at 17.9 min. and the negative signals are assumed to be system peaks, which occur due

to aging of THF in the prepared samples and in the mobile phase.

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PLGel Mixed 2 columns

PS 1990 g/mol

PS 19880 g/mol

Chapter 4: Results & Discussion

30

Figure 17: Calibration curves of PLGel Mixed double column, created with PMMA and PS

standards.

Figure 17 shows the calibration curves of both the PMMA calibration and the PS calibration.

As can be seen from this figure, both calibration curves show a linear trend after the permeation

limit corresponding to the SEC region of the curve, up to a molar weight of about 107 g/mol

and are quite comparable. This result was expected due to the fact that even though PMMA

and PS have different hydrodynamic sizes, the retention mechanism is for both standards the

same in SEC. From these calibration curves it can be concluded that both PS and PMMA can

be used to determine PLGA molecular weights under these conditions.

Table 2: PLGA molecular weight results calculated according to PMMA and PS calibration

curve fitting given in Figure 17.

Calibration PLGA 21 kDa PLGA 10.5 kDa

PMMA 5758.52 g/mol 11005.14 g/mol

PS 5733.71 g/mol 10751.71 g/mol

0

1

2

3

4

5

6

7

8

0 5 10 15 20

Log

MW

VR (mL)

PMMA & PS calibration curves of PLGel Mixed 2 columns

PMMA Calibration

PS Calibration

Chapter 4: Results & Discussion

31

In Table 2, the results are given of the molecular weight calculations with the injected PLGA

samples of 21 kDa and 10.5 kDa, using a third order fitting. As can be concluded from this

table, the calculations for PLGA with a molar weight of 10.5 kDa are fairly accurate, while the

calculation results for PLGA with a molar weight of 21 kDa show largely different numbers

than what was expected.

There are multiple scenarios that could explain the results, the PLGA corresponding to

the molar weight of 21 kDa could have decayed, which would have resulted in a lower

molecular mass and is a feasible explanation due to the biodegradable nature of PLGA.20 The

other scenario is that the PS and PMMA calibrations were not representative for the PLGA

molecules due to the different ways that the molecules fold, although this case is less likely

because of the fact that both calibrations give nearly the same results. Another potential issue

could be that the PLGA of 21 kDa was labeled incorrectly, or that the PLGA sample

corresponding to the 21 kDa molecular weight was deposited into the wrong vial, which could

explain the higher viscosity of the PLGA which was labeled to be the 10.5 kDa PLGA (as

higher molecular weight polymers generally have a higher viscosity than lower molecular

weight polymers).5, 21

Chapter 4: Results & Discussion

32

4.2 Separation of PLGA mixture with experimental core-shell columns

4.2.1 Column efficiency determination with PLGA

Figure 18: Chromatogram of PLGA mixture, blank THF sample and toluene marker,

separated with the Phenomex core-shell B109 column.

Figure 18 depicts the chromatogram of the 10.5 kDa and 21 kDa PLGA mixture, as well as the

toluene and blank sample. As is shown in the chromatogram, there are broad signals visible at

6.03 and 6.76 min., which are assigned to the PLGA mixture due to the absence of these signals

in the blank sample. The signal which is shown as the PLGA and blank sample at 7.14 min., as

well as the negative peaks are assumed to be system peaks, caused by the aging of THF. The

signal at 7.00 min. is assigned to the toluene marker, as it corresponds to the predicted

permeation limit calculated by Eq. (4). The signal at 7.39 min. is expected to be either an

impurity in the toluene sample or caused by aging of the sample, as the toluene sample was

prepared from a different THF bottle than the blank and PLGA samples, and can therefore

show different signals.

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Phenomenex core-shell B109

Blank

t0

PLGA

Chapter 4: Results & Discussion

33

Figure 19: Chromatogram of the PLGA mixture and individual PLGA injections, separated

with the Phenomenex core-shell B109 column.

Figure 19 shows the chromatogram of the PLGA mixture and individual PLGA injections, with

fixed y- and x-axis at the PLGA signals. There is a good separation visible between the 10.5

kDa and 21 kDa-labeled PLGA samples at 6.03 and 6.76 min. It has been determined that these

signals indeed correspond to the used PLGA samples, by comparing them to the separate

injections of both polymers. It can be observed that baseline separation had not been realized,

but the Phenomenex core-shell B109 column achieves the highest resolution from all

investigated columns, which can be related to the fact that the stationary phase consisted of

small core-shell particles, resulting in lower chromatographic dispersion and thus higher

resolution.8 Also the flowrate may have a role in the different selectivity, as a flowrate of 0.2

mL/min was used, in contrast to the 1.0 mL/min flowrate that was used for the measurement

with the PLGel columns and which can potentially reduce the plate height according to Eq. (3),

depending on the effective constants.

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1400

1900

2400

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3400

3900

5,4 5,6 5,8 6 6,2 6,4 6,6 6,8 7

Inte

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Time (min)

Phenomenex core-shell B109

PLGA 10.5 kDa

PLGA 21 kDa

PLGA Mixture

Chapter 4: Results & Discussion

34

4.2.2 SEC calibration with polymethylmethacrylate

Figure 20: Calibration curve of core-shell B109 column, created with PMMA standards.

Figure 20 shows the calibration curve of the Phenomenex core-shell B109 column with PMMA

standards using the HPLC setup with the RID. As can be seen from the calibration curve, there

is a linear region between the permeation limit and the molar weight of 104.4 g/mol, and a non-

linear region afterwards, which might indicate the exclusion limit in which HDC effects occur.

This HDC region is clearly visible on the calibration curve for this column with the used

standards due to the small particles (98 Å poresize) in the stationary phase. Using a third order

fitting, the PLGA sample sizes were subsequently calculated, and given in Table 3 below.

Table 3: PLGA molar weight calculated according to the PMMA calibration curve fitting

given in Figure 20.

Calibration PLGA 21 kDa PLGA 10.5 kDa

PMMA 600.67 g/mol 5996.6 g/mol

y = -34,306x3 + 131,7x2 - 174,44x + 82,699R² = 0,997

0

1

2

3

4

5

6

7

0,8 0,9 1 1,1 1,2 1,3 1,4

Log

MW

VR (mL)

PMMA calibration curve of Phenomenex core-shell B109 column

PMMA Calibration

Poly. (PMMA Calibration)

Chapter 4: Results & Discussion

35

As can be seen from this table, the calculated molar weight distributions are not in line with

the expected values, as both the PLGA of size of 21 kDa and 10.5 kDa have significant lower

calculated molar weights, even though the samples have molecular weights that fall within the

linear region of the calibration curve. The results do show that the 10.5 kDa PLGA has a higher

molecular mass compared to the 21 kDa PLGA, which is in agreement to the results given in

Table 2 and might be an indication that the 21 kDa PLGA had been substantially decayed. The

unexpected calculated values can also be the consequence of the fact that the PMMA molecules

have a different way in which they fold compared to PLGA in this column with sub-3 µm

particles. A proposed solution would be to use a different set of calibration standards, as this

result shows that PMMA is not capable of providing a calibration curve that is accurate.

4.2.3 SEC Calibration with 3 coupled columns

Figure 21: Calibration curve of core-shell B111, B104 and B109 mixed columns, created

with PS standards.

Figure 21 depicts the calibration curve of the 3 core-shell B111, B104 and B109 mixed

columns. As can be seen from this figure, multiple areas in the curve can be distinguished.

y = -0,9051x3 + 9,6288x2 - 36,563x + 52,733R² = 0,9896

0,00

1,00

2,00

3,00

4,00

5,00

6,00

7,00

2,4 2,6 2,8 3 3,2 3,4 3,6 3,8 4

Log

MW

VR (mL)

PS calibration curve of Phenomenex core-shell B111, B104 and B109 columns

PS Calibration with Slalom

PS Calibration

Poly. (PS Calibration)

Chapter 4: Results & Discussion

36

There is a linear range between a molecular weight of 103.3 g/mol and 104.3 g/mol, after which

an elevation of the calibration curve occurs that continues in a linear fashion between 105.5

g/mol and 106.1 g/mol. These different ranges are caused by the combining of columns, these

stationary phases contain different particle and pore sizes, which has the same effect as a long

mixed column. It is expected that the linear molecular weight ranges are separated using a SEC

mechanism. Above the second molecular weight range, SC is clearly observed which is related

to the stretching of the higher molecular weight polymers in the stationary phase.9 Furthermore,

the data was fitted using a 3th order curve and is fairly precise when leaving out the SC region.

It can be concluded that this set of columns are efficient at separating low-molecular weight

polymers up to a molecular weight of 106.1 g/mol, using a SEC mechanism, but might show

inaccuracy at the elevation area.

4.2.4 Analysis of large polydispersity PS

Figure 22: Chromatogram of large polydispersity PS, blank THF sample and toluene marker,

separated on three Phenomenex core-shell columns (B111, B104 and B109).

Figure 22 depicts the chromatogram of a large polydispersity PS sample, blank and toluene

marker.

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Phenomenex core-shell B111, B104, B109

Blank

Toluene

PS Broad

Chapter 4: Results & Discussion

37

As can be seen from this chromatogram, the PS sample shows a large distribution which is

characteristic for large polydispersity polymers, and is absent from the blank sample which

indicates that this is indeed the polystyrene sample. It was expected beforehand that due to the

molecular weight distribution overlap with the elevation of the calibration curve at a molecular

weight of 105.2 g/mol, the peak would elute in a non-Gaussian shape. This result shows that

this is not the case, which was unexpected.

To induce this non-Gaussian peak shape, a larger polydispersity polystyrene might need

to be used. Furthermore, it can be seen that during the polystyrene injection, residue of the

toluene sample is injected, which can be solved by running a longer needle wash.

4.2.5 Concluding remarks regarding SEC analysis

From all the SEC measurements performed, it can be concluded that the proposed method with

the respective double columns and core-shell columns are indeed capable of separating the used

PLGA samples, but to be able to make a definite conclusion it is necessary to use undegraded

PLGA building blocks to validate the SEC method and apply these building blocks to prepare

nanoparticles with known compositions. The focus will now go to HDC analysis of polystyrene

samples.

Chapter 4: Results & Discussion

38

4.3 HDC Measurements

Figure 23: Chromatogram of 3-NBS acid separated on an Agilent PL-PSDA Cartridge Type 1

column.

To test the performance of the used HDC stationary phase, column tests have been performed

with 3-NBS acid, uracil and potassium dichromate. Figure 23 depicts the chromatogram of 3-

nitrobenzenesulfonic acid (3-NBS acid) separated on an Agilent PL-PSDA Cartridge Type 1

HDC column, performed under the same conditions as the column test given by the

manufacturer, shown in Appendix 1. As can be seen from this chromatogram, there is a broad

peak visible with a retention time at 3.732 min.

Furthermore, substantial tailing could be observed of several minutes, which is unexpected

when compared to the column performance test supplied by the manufacturer which shows a

sharp peak with no tailing. According to the predicted value of V0 calculated with Eq. (4), from

which the t0 marker retention time is deduced, it can be seen that the expected elution time is

7.2 min. (using a porosity of 0.42). This calculation is an approximation, as the practical elution

time given by the manufacturer is 8.7 min. Nevertheless, it can be concluded that the column

performance test results conducted in this study are not in line with the column test results from

the manufacturer.

Figure 24 and Figure 25 show the injection with uracil and potassium dichromate on the same

column, which are general t0 markers in HDC.

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9

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Inte

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Agilent PL-PSDA Cartridge Type 1

3-NBS Acid

Chapter 4: Results & Discussion

39

Figure 24: Chromatogram of uracil separated on an Agilent PL-PSDA Cartridge Type 1

column, with a flowrate of 1.6 mL/min and retention time at 3.77 min.

Figure 25: Chromatogram of potassium dichromate separated on an Agilent PL-PSDA

Cartridge Type 1 column, with a flowrate of 1.7 mL/min and retention time at 2.86 min.

As can be seen from both chromatograms of uracil and potassium dichromate injections on the

HDC column, both signals show broad signals with substantial tailing. Uracil has a retention

time of 3.77 min. at a flowrate of 1.6 mL/min, while potassium dichromate has a retention time

of 2.86 min. at a flowrate of 1.7 mL/min, which are early elution times for t0 markers on this

column, calculated under the respective conditions according to Eq. (4).

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Agilent PL-PSDA Cartridge Type 1

Uracil

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Agilent PL-PSDA Cartridge Type 1

Potassium dichromate

Chapter 4: Results & Discussion

40

Figure 23-25 therefore suggest that the column stationary phase had been degraded. A possible

explanation could be that the stationary phase was damaged during column conditioning.

It can be concluded that the available Agilent PL-PSDA Cartridge Type 1 column

could not be used for the analysis of polystyrene analysis. This issue could be resolved by

using a replacement of this HDC column.

Chapter 5: Conclusion

41

5. Conclusion

With the analysis of PLGA on the different SEC columns investigated, insight was gained on

the various column efficiencies. It can be concluded that some columns are indeed more

effective for the separation of the PLGA mixture with labelled 10.5 kDa and 21 kDa molar

weight distributions. Especially the PLGel Mixed double column and the Phenomenex core-

shell column showed good separation results, where both signals in the mixture could be clearly

distinguished. When the retention times of the PLGA samples were related to the calibration

curves prepared with PS or PMMA standards, the PLGel Mixed double column showed

accurate molecular weight results for the 10.5 kDa PLGA sample, while the molecular weight

calculation performed with the Phenomenex core-shell column gave results that were

inaccurate for both 21 kDa and 10.5 kDa PLGA samples. The latter column can therefore not

be used for molecular weight determinations under the conditions that are applied in this study.

The high efficiency of the Phenomenex core-shell column can furthermore be related to the

smaller particles in the stationary phase which decreases the plate height and therefore

increases the resolution.

Moreover, it can be concluded that the PLGA with a molecular weight of 21 kDa had

been degraded, which was confirmed by SEC molecular weight determination measurements

with the PLGel Mixed double column and the Phenomenex core-shell column, while it shows

that the 10.5 kDa PLGA sample still had the original molecular weight distribution. In this

study it is also shown that there is no significant difference between using PS or PMMA as

calibration standards, as the calibration curves resulting from injections with either of the

polymers were similar.

To validate the proposed second dimension SEC methods for the comprehensive

analytical MAnIAC system, HDC was performed with the Agilent PL-PSDA Cartridge Type

1 column, but as shown during the column test with 3-NBS acid, uracil and potassium

dichromate, the performance of this column was insufficient due to the extensive tailing of

peaks and retention times that did not correspond to the specifications of the manufacturer.

Further HDC measurements could therefore not be conducted with this column to validate the

SEC methods which were developed in this study.

Chapter 6: Future Prospects

42

6. Future Prospects

A priority in this study was testing out the right columns and conditions in which polymer

mixtures can effectively be separated by the SEC mechanism. These findings can subsequently

be used in the optimization of the envisaged comprehensive analytical system for the analysis

of polymers. It is expected that especially the application of Phenomenex core-shell columns

are a good choice for this system, due to the high flowrates that can be utilized which facilitates

faster separations in the second dimension. Furthermore, the Phenomenex core-shell column

proved to give the highest resolution of all the tested columns, despite the smaller void volume

of core-shell columns compared to columns with entirely porous particles. For future work, it

would be viable to use the 3 coupled core-shell columns to inject the PLGA samples, and

determine its molecular weight distributions with undegraded PLGA building blocks.

An important stage in the development of the second dimension is the validation of the

method. It was envisaged to perform this validation with HDC, but due to issues with the

stationary phase of the HDC column, this was not performed. In future studies, it is advised to

continue the planned validation with HDC, to see if the method is indeed capable of separating

polymer nanoparticles with known polymer lengths and compositions.

An extension of this work would be investigating the use of RPLC or IEC columns

instead of the SEC separation methods studied in this research. Using RPLC or IEC columns,

other characteristics of polymers like hydrophobicity and charge can be determined.

The next step in the development of the comprehensive analytical system would be

combining both first and second dimension separation methods. To do this, HDC and SEC

conditions need to be optimized for both methods to achieve the best possible performance.

References

43

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Appendix

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Appendix 1: Column performance report of Agilent PL-PSDA Cartridge Type 1 column