TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2013 Waters Corporation

Figure 5. Effect of Flow Rate on Polystyrene Mp 474 Da and

Polystyrene Mp 2.92 kDa Using an ACQUITY APC 45 XT 1.7 µm Column.

AU

0.000

0.010

0.020

Minutes

5.0 6.0 7.0 8.0 9.0

AU

0.000

0.010

0.020

Minutes

1.3 1.5 1.7 1.9 2.1 2.3

Minutes

4.0 5.0 6.0 7.0 8.0

Minutes

0.9 1.1 1.3 1.5 1.7 1.9

Polystyrene Mp 474 Da PDI 1.16 Polystyrene Mp 2.92 kDa PDI 1.06

Flow Rate: 0.2 mL/min

Flow Rate: 0.8 mL/min

Flow Rate: 0.2 mL/min

Flow Rate: 0.8 mL/min

DISCUSSION

Traditional styrene-divinyl benzene gels come in a variety of pore sizes which gives them the ability to analyze samples having a wide range of molecular weights. Rigid hybrid materials with pore sizes of 45Å, 125Å, 200Å, and 450Å (Figure 1) were prepared with the intent of having comparable usable molecular weight ranges as the commercially available HSPgel HR

materials (Tables 1 and 2). The calibration curves (Figures 2 and 3) demonstrate that the usable molecular weight ranges for the APC XT materials and the HSPgel HR materials are comparable; 100 Da to 400 kDa.

Squares of the correlation coefficients (R2) and slopes were calculated for the log Mp versus elution volume for the linear region of each of the curves (Table 4). The R2 values generated for the APC XT materials were comparable to those obtained on the HSPgel HR materials.

In addition to the R2 value, the slope of the linear region was also determined. The slope of the calibration curve is an indicator of the separation range

of the column. A steeper slope provides a broader molecular weight separation range, but less resolution. A shallower slope has a narrower separation range, but more resolution. The slopes calculated for the single ACQUITY APC XT columns are 3-4X shallower than the slopes calculated on the columns packed with HSPgel HR materials; more resolution in a narrower separation range. One common practice to increase the separation range is to use multiple columns of different pore sizes.

The choice of flow rate is at times a compromise between resolution, analysis time, and column operating limits. The recommended maximum flow rate for the HSPgel HR 3µm columns was 0.6 mL/min. The scaled flow rate of 0.35 mL/min was easily achieved on the APC XT materials and could be exceeded. From the

flow rate study using the APC XT 45 1.7 µm material, the highest column efficiencies are achieved at flow rates greater than 0.5 mL/minute (Figure 4). This can be visualized by the increased resolution of those molecular weights in the polystyrene oligomer with the peak molecular weight of 474 (Figure 5). Polystyrene with a peak molecular weight of 2.92 kDa was analyzed at 0.2 mL/min and 0.8 mL/min to determine the Mw, Mn, and Mp values. They were comparable to the values given for the standard and not significantly different at the two flow rates (Table 5).

INTRODUCTION

The technique of Gel Permeation Chromatography (GPC) was first described by J.C. Moore1 for the fractionation of synthetic polymers by molecular weight using crosslinked polystyrene gels in non-aqueous mobile phases. The separation mechanism for GPC is based on the size of the polymer in solution relative to the pore size distribution of the

column’s packing material. This separation mode relies on the absence of all other separation mechanisms, such as hydrophobic interaction or ion-exchange.

Historically, packing materials for GPC have been largely organic polymers, such as hydrophobic styrene-divinyl benzene gels or hydrophilic methacrylate gels. However, these organic polymer particles do not provide the highest efficiency due to their lack of rigidity and susceptibility to compressing under pressure.

The goal of this investigation was to evaluate the use of low dispersion chromatographic systems in combination with high efficiency essentially non-

compressible hybrid organic / inorganic packing materials. These hybrid materials have been synthesized to have pore sizes ranging from 45Å to 450Å, and were evaluated with regard to their ability to perform size-based separations. Initial evaluations were performed to determine the usable molecular weight range for each of the pore size materials and the results are compared to those of commercially available GPC materials of similar pore size.

APPLICABILITY OF RIGID HYBRID-BASED PACKING MATERIALS FOR SIZE-BASED SEPARATIONS OF SYNTHETIC POLYMERS

B. Alden, E.S.P. Bouvier, P. Iraneta, N. Lawrence, M. Savaria, J. Shia, M. Summers, T. Walter, J. Wilson, K. Wyndham

Waters Corporation 34 Maple Street Milford MA 01757 USA

METHODS

A. Materials Evaluated

The hybrid packing materials had pore sizes ranging from 45Å to 450Å. The 125Å, 200Å, and 450Å materials were 2.5 µm, and the 45Å material was 1.7 µm. These materials were evaluated in 4.6 x 150 mm stainless steel hardware.

HSPgel™ HR columns (6.0 x 150 mm) were chosen as the commercially available product that would possess a similar pore size range as the hybrid materials. All HSPgel HR materials are 3 µm styrene-divinyl benzene gels.

B. Chromatographic Conditions

System: Prototype ACQUITY APC System using an ACQUITY TUVe with a 5 mm stainless steel flow cell (1500 nL).

System variance2 ( 2): 2.70 µL2

Detection Wavelength: 254 nm

Column Temperature: 30 °C

Mobile Phase: 100 % tetrahydrofuran (THF)

Injection Volume: 2.0 µL (4.6 x 150 mm) 3.4 µL (6.0 x 150 mm)

References

1. J. C. Moore; Gel Permeation Chromatography. I. A New Method for

Molecular Weight Distribution of High Polymers. J. Polymer Science:

Part A 1964, 2, 835-843

2. Striegel, Andre M., et al. Modern Size-Exclusion Liquid Chromatography

Practice of Gel Permeation and Gel Filtration Chromatography. 2nd ed.

Hoboken, NJ: John Wiley and Sons, Inc. 2009

A. Calibration Curves: 0.6 mL/minute

CONCLUSION

ACQUITY APC XT materials with 45Å, 125Å,

200Å, and 450Å pores, demonstrated a separation range of 200 Da to 400 kDa for linear polystyrenes; comparable to the commercially available styrene-DVB gel family.

The benefits of using a low dispersion

ACQUITY APC system with 4.6mm ID columns packed with rigid hybrid particles for sized-based separations are:

- Reduction in analysis times.

- Lower consumption of solvents.

- Maintenance of resolution for oligomers.

- Accurate determination of Mw values.

Size-based separations using aqueous mobile

phases have been achieved using ACQUITY APC AQ materials based on the same rigid, hybrid particles (Figure 6).

100

1,000

10,000

100,000

1,000,000

10,000,000

0.25 0.45 0.65 0.85

Poly

styr

ene

Mo

lecu

lar

Wei

ght (

Da)

Elution Volume (mL): Normalized to Column Volume

HSPgel HR 4.0

HSPgel HR 3.0

HSPgel HR 2.5

HSPgel HR 2.0

HSPgel HR 1.0

Figure 2. HSPgel HR Columns: Calibration Curves Generated

Using Linear Polystyrene Standards.

100

1,000

10,000

100,000

1,000,000

10,000,000

0.25 0.45 0.65 0.85

Poly

styr

ene

Mo

lecu

lar

Wei

ght (

Da)

Elution Volume (mL): Normalized to Column Volume

ACQUITY APC XT 450

ACQUITY APC XT 200

ACQUITY APC XT 125

ACQUITY APC XT 45

Figure 3. ACQUITY APC XT Columns: Calibration Curves

Generated Using Linear Polystyrene Standards.

Table 1. ACQUITY APC™ XT Materials

Molecular Weight Range

APC XT 45 1.7µm 200-5,000

APC XT 125 2.5µm 1,000-30,000

APC XT 200 2.5µm 3,000-70,000

APC XT 450 2.5µm 20,000-400,000

Table 2. HSPgel HR Materials

Molecular Weight Range

HR 1.0 3µm 100-1,000

HR 2.0 3µm 500-10,000

HR 2.5 3µm 1,000-20,000

HR 3.0 3µm 2,000-60,000

HR 4.0 3µm 10,000-400,000

Table 3. Linear Polystyrene Standards Prepared in 100 % THF

Ready-Cal Polystyrene Kits;

2,520,000 - 474 Da 66,000 - 266 Da

Mw > 1 M [0.5 mg/mL] Mw < 1 M [1.0 mg/mL]

Polystyrene 370 Da Polystyrene 266 Da Polystyrene 162 Da

1.0 mg/mL 1.0 mg/mL 1.0 mg/mL

B. Flow Rate Study

A study was performed to assess the influence of flow rate on size-based separations. Higher efficiencies were achieved for the monomer, dimer, and trimer of polystyrene as flow rate increased. A higher molecular weight polystyrene was not as influenced.

Figure 4. Flow Rate Study Using ACQUITY APC XT 45 1.7 µm.

Figure 1. SEM Images Highlighting the Morphology Changes

for the Different Pore Sizes.

45Å 125Å

200Å 450Å

RESULTS

0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

40,000

45,000

0.00 0.20 0.40 0.60 0.80 1.00 1.20

4Ef

fici

ency

Flow Rate (mL/min)

Polystyrene Mp 162 PDI 1.00 Polystyrene Mp 266 PDI 1.00

Polystyrene Mp 370 PDI 1.00 Polystyrene Mp 2,920 PDI 1.06

Table 4. Calibration Curve Data for Linear Region

R2 Slope

APC XT 45 0.998 -2.58

APC XT 125 0.996 -1.71

APC XT 200 0.989 -1.66

APC XT 450 0.998 -1.48

HSPgel HR 1.0 0.997 -5.32

HSPgel HR 2.0 0.981 -8.46

HSPgel HR 2.5 0.992 -7.40

HSPgel HR 3.0 0.997 -6.88

HSPgel HR 4.0 0.996 -6.53

= Linear Region

= Linear Region

Table 5. Molecular Weight Values for Polystyrene Standard Mp 2920 Da

Mw Mn Mp PDI

Actual 2870 2720 2920 1.06

0.2 mL/min 3038 2853 3173 1.06

0.8 mL/min 2974 2791 3075 1.07

Figure 6. Pullulan Standards on ACQUITY APC AQ 200 2.5µm

100 mM NaNO3 in 80:20 H2O/acetonitrile (v/v), Flow Rate: 0.6 mL/min, Column Temperature: 40 °C.

µRIU

0.00

10.00

20.00

Minutes

1.00 2.00 3.00

107K

47K

21K 9

.6K

6.1

K

4X Faster

With

Better

Resolution