Method conditions

Excellent resolution and fast run times2 x OligoPore, 4.6 x 250 mm columns gave excellent oligomeric resolution for the PS 580 sample. Resolution was largely maintained at the 1.2 mL/min flow rate, where run time was less than 6 minutes.

Figure 1: PS 580 run on 2 x OligoPore, 4.6 x 250 mm, columns at 0.3 (bottom trace), 0.6 (middle trace), and 1.2 mL/min (top trace). Normalized retention time.

2 x MesoPore 4.6 x 250 mm, columns gave excellent resolution for the oligomers in epoxy resin samples. Some exclusion was observed for higher molecular weights.

Figure 2: Epikote 1004 (bottom trace), 1007 (middle trace) and 1009 (top trace) run on 2 x MesoPore, 4.6 x 250 mm, columns at 0.3 mL/min.

Again, this resolution was largely maintained at the highest flow rate – and run time of less than 6 minutes.

Figure 3: Epikote 1004 run on 2 x MesoPore, 4.6 x 250 mm, columns at 1.2 mL/min.

Additional reduction in solvent consumptionThe lower flow rate required for GPC/SEC columns with an internal diameter of 4.6 mm gives a 70% saving in solvent consumption compared to 7.5 mm id. If the column internal diameter is reduced to 2.1 mm this saving becomes 94%. As is the case with UHPLC, using 2.1 mm id columns careful control of system dispersion, which includes the detector flow cell volume, is critical.

Resolution was achieved with 2 x MesoPore, 2.1 x 250 mm, columns at all flow rates. This resolution could be improved by further reduction of system dispersion volume.

Figure 4: Epikote 1004 run on 2 x MesoPore 2.1 x 250 mm, columns at 0.06 (bottom trace), 0.12 (middle trace), and 0.24 mL/min (top trace). Normalized run time.

Investigating Miniaturization in GPC/SEC

Stephen Luke, Peter Cooke, and Graham Cleaver 5991-5983ENAgilent Technologies, UK

Introduction

Conclusions

Experimental

Results and Discussion

Results and Discussion

2015

Parameter Change Benefit Consideration

Flow rate LowerDecreased

solvent consumption

Pump flow rate precision

Peak volume LowerIncreased sensitivity

System dispersion

volume

Peak width NarrowerHigher

resolutionDetector data

(sampling) rate

Sample Columns

PS 5802 x Agilent OligoPore, 4.6 x 250 mmLinear MW range up to 3,300 g/mol

Sample ColumnsEpikote 1004Epikote 1007Epikote 1009

2 x Agilent MesoPore, 4.6 x 250 mmLinear MW range up to 25,000 g/mol

Column Set 0.3 mL/min 0.6 mL/min 1.2 mL/min

2 x OligoPore, 4.6 x 250 mm

28 bar 56 bar 112 bar

2 x MesoPore, 4.6 x 250 mm

41 bar 82 bar 170 bar

Column Set0.06

mL/min0.12

mL/min0.24

mL/min

2 x OligoPore, 2.1 x 250 mm

21 bar 42 bar 84 bar

2 x MesoPore, 2.1 x 250 mm

30 bar 20 bar 125 bar

Experimental

Column: 4.6 x 250 mm 2.1 x 250 mm

Run TimeMobile phase:

THF

Flow rate:0.3 mL/min0.6 mL/min1.2 mL/min

0.06 mL/min0.12 mL/min0.24 mL/min

24 minutes12 minutes6 minutes

Sample conc.:

1 mg/mL

Inj. volume: 2 μL 0.5 μL

Temperature:

50 °C

Sample Columns

PS 5802 x Agilent OligoPore, 2.1 x 250 mmLinear MW range up to 3,300 g/mol

Sample Columns

Epikote 1004 2 x Agilent MesoPore, 2.1 x 250 mmLinear MW range up to 25,000 g/mol

GPC/SEC is an important liquid chromatographic technique for determining the molecular weight distribution and averages of a polymer and for comparing batch-to-batch polymer quality.

Miniaturization, the use of smaller column dimensions, has been a popular approach in many liquid chromatographic techniques. The benefits of miniaturization include reduced solvent costs, higher throughput, increased detector response, and taking full advantage of the latest advances in liquid chromatography instrument design.

In this work we investigate how miniaturization can be applied to gel permeation chromatography, discuss critical considerations, and determine what benefits the approach brings for size based separations.

MiniaturizationConventional analytical-scale GPC/SEC separations are performed using columns with a length of 300 mm and internal diameter between 7.5 and 8.0 mm. Optimum linear velocities in GPC are relatively low and as a result flow rates of 1.0 mL/min are used. To achieve acceptable resolution, multiple columns are often connected in series leading to typical run times of 45 to 60-minutes per analysis with solvent consumption of up to 60 mL.

The impact of GPC/SEC miniaturization is summarized below.

GPC/SEC columns with a length of 250 mm and an internal diameter of 4.6 mm, for example PLgel MiniMIX, have been available for some time. This work investigates what is now possible with high performance GPC/SEC columns, new column formats, and the latest advances in liquid chromatography instrument design.

GPC/SEC instrumentsAn Agilent 1260 Infinity GPC/SEC system with 1290 Infinity II µRID was used.

The Agilent 1260 Infinity Isocratic Pump features virtually pulse-free and stable solvent flow, making it the ideal pump for GPC/SEC applications where run-to-run and day-to-day precision of retention times is important.

The 1290 Infinity II µRID is optimized for use with fast separations on narrow internal diameter columns, optimized fluidic connections, 2.1 µL flow cell volume, and a maximum data rate of 148 Hz.

Data were collected and processed using Agilent GPC/SEC software version 1.2.

High performance GPC/SEC columnsHigh performance columns that deliver significantly increased resolution compared to a conventional GPC/SEC column set allow the use of fewer columns in series and higher linear velocities, without sacrificing separation quality.

To clearly demonstrate the performance of miniaturization we used polystyrene and epoxy resin samples with columns that would resolve numerous oligomer peaks.

Further miniaturization and faster separationsWe also investigated the use of further miniaturization with even narrower internal diameter columns.

To investigate how fast miniaturized GPC/SEC separations can be, each sample was run at three different flow rates; from the normal linear velocity (equivalent to 1.0 mL/minute on a 7.5 mm id column) to 4x this flow.

Reproducible and reliable resultsReproducible retention times are critical for accurate and reliable GPC/SEC. 2 x MesoPore 2.1 x 250 mm, columns show retention time precision of 0.04% RSD even at the lowest flow rate of 0.06 mL/min.

Figure 5: PS 580 run six times on 2 x OligoPore, 2.1 x 250 mm, columns at 0.06 mL/min.

The PS/DVB particles packed in OligoPore and MesoPore high performance columns are a proven technology for GPC/SEC, meaning that methods can be transferred with confidence without the risk of absorbance effects or other interactions between the analytes and stationary phase.

High pressure rating not requiredAs discussed, GPC/SEC miniaturization does require the careful control of system dispersion volume that is a feature of UHPLC instruments. However, it does not require the very high pressures that these instruments are designed to support. Pressures generated by the different column/flow rate combinations were all well within the 600 bar pressure limit of the Agilent 1260 Infinity GPC/SEC system.

Table 1: Pressures at each column/flow rate combination

In combination with a low volume detector flow cell and high detector data rate, miniaturized high performance GPC/SEC columns are capable of generating excellent resolution and fast run times.

Miniaturization to high performance GPC/SEC columns with a 2.1 mm internal diameter allows solvent consumption to be reduced by 94% compared to 7.5 mm columns.

Providing high precision solvent delivery is available at low flow rates, highly reproducible results can be obtained with miniaturized high performance GPC/SEC columns.

Miniaturized high performance GPC/SEC columns packed with proven PS/DVB particles do not require very high pressure to operate even at elevated flow rates and thus, provided that other dependencies discussed are met, can be used on existing instruments.