richard lentner comprehensively better! applications ... · effect of injector loop size on...

Richard Lentner

Applications Specialist

GPC/SEC Instrumentation

March 26, 2013

Comprehensively better!

Increase performance

for your polymer analysis

A Typical GPC/SEC System

A Typical GPC/SEC System: Solvent Choice

Sample Preparation for GPC

Criteria for Solvent Selection

True sample solubility to avoid non-size exclusion effects

Compatibility with columns

Permit adequate detection (eg refractive index, UV cut off)

Safety (eg toxicity, elevated temperature, etc)

Use an aliquot of the eluent to prepare the solution

Sample concentration depends on molecular weight

For polymers, avoid high shear stirring

Dissolution time and temperature will depend on molecular weight

and crystallinity of polymer

Filter samples to remove insoluble material (0.5 - 1.0um filters)

Typical Range of Solvents used in GPC

5

• A wide range of solvents are

used in GPC with very varied

viscosities

• Elevated temperature helps to

reduce the viscosity of these

solvents improving column

lifetime

A Typical GPC/SEC System: Loading

Sample Loading for GPC

General Guidelines

viscosity = MW * concentration

For high MW samples use lower concentration and if detector

response requires it, increase injection volume

For low MW samples use higher concentrations and avoid larger

injection volumes to maintain high resolution

MW Conc (%) Inj vol (ul)

<50,000 0.20-0.50 20-50

50,000 - 500,000 0.10-0.20 50-200

>500,000 .01-0.10 50-200

Effect of Concentration on Peak Shape

and Resolution

Column: PLgel 10µm MIXED-B

300x7.5mm

Eluent: THF

Flow Rate: 1.0ml/min

Detector: UV

Polystyrene standards

1. 8,500,000 4. 34,500

2. 1,130,000 5. 5,100

3. 170,000 6. 580

0.05% 0.10%

0.15% 0.20%

Effect of Injector Loop Size on Resolution

9

Column : PLgel 3 µm MIXED-E

300 x 7.5 mm

Eluent : THF

Flow rate : 1.0 ml/min

Sample : Epikote 1001

epoxy resin

20 µl loop

200 µl loop

Injection loop is a major

contribution to system dead

volume, use reduced injection

volume and increase

concentration to maintain

sensitivity

1260 Infinity Autosampler

The Agilent 1260 Infinity Standard Autosampler provides reliable injections from 0.1 μL to 100 μL covering micro and analytical scale applications.

Sample carryover is non-existent, ensuring reliable results.

The ALS is easily adaptable to increase injection volumes for semi-preparative GPC.

Agilent 1260 Infinity GPC/SEC System

10

Key Features Benefit

New Needle seat • Improved lifetime

Injection Volume:

0.1 – 100 µl (multi-draw 1500ul) or

0.1 – 900 µl (multi-draw 1.8 ml)

• Largest injection volume range for single stroke

injections from analytical to semi-prep

Very low AFR • High robustness proven by 1000ds of installations

2 ml and 6 ml vials • Supports any routine vial-based workflow

A Typical GPC/SEC System: Flow Precision

Effect of Flow Rate on Resolution

12

• Flow rate strongly affects

resolution

• Every column has an

optimum flow rate

• In GPC the mass transfer

effect is prominent

13

Eluent : THF

Columns : PLgel 100Å

Test probe : ODCB

Optimum flow rate may differ

with column particle size

Flow Rate and Efficiency

Affect of Pump Flow rate in GPC Results

14

A small change in flow rate can have a large effect on GPC molecular

weight results

1260 Infinity Isocratic Pump


15

Feature Detail Benefit

Wide flow range 0.2 to 10 mL/min for 4.6, 7.5

and 25 mm ID columns

Covers micro, analytical

and prep-GPC applications

Flow precision ≤ 0.07 % RSD, or ≤ 0.02 min

SD whichever is greater

Excellent precision and

reproducibility

Low operating &

maintenance costs

proven robust design & material

for long instrument uptime

Low cost of ownership

Pressure pulsation < 2 % amplitude (typically < 1.3

%), or < 0.3 MPa (3 bar)

whichever is greater

Better chromatography and

essential when using

refractive index detection

Highest

performance for

isocratic LC

600 bar is the new HPLC

standard

System working well within

operating range – no stress

to system

Flexible and

upgradable

Mix and match modules,

complementary detection

technologies

Maximize the applications

covered. Make full use of

all modules in laboratory.

1260 Infinity Degasser

Many common GPC/SEC solvents outgas

• Important to thoroughly degass solvent

• RID especially susceptible

Agilent 1260 Infinity Degasser: reduces baseline noise due to

high degassing capacity

Agilent 1260 Infinity Standard Degasser Performance Specifications

Maximum flow rate 10 mL/min per channel

Number of channels Four

Internal volume per channel Typically 12 mL per channel

Materials in contact with solvent PTFE, PEEK

pH range 1 – 14

Excellent Reproducibility

New level of molecular weight data precision


17

The excellent flow

precision of the Agilent

1260 Infinity Series pump

ensures the ultimate in

reproducible molecular

weights

Why use Elevated Temperature?

19

GPC applications employing elevated temperature generally

fall into three categories :

1. To reduce solvent viscosity for improved mass transfer and

chromatographic separation

2. To reduce system pressure and column damage

3. To provide a stable thermal environment for detectors

4. To achieve and maintain sample solubility

Effect of Temperature on Separations on Viscous

Solvents

20

Column : PLgel 5 µm MIXED-C

300 x 7.5 mm

Eluent : DMF

Flow rate : 1.0 ml/min

PEO/PEG standards

990,000 252,000

86,000 18,000

4,800 200

Increased temperature :

Reduced operating

pressure

Improved resolution,

particularly at high MW

Effect of Temperature on Column Pressure

21

Column PLgel 5 µm MIXED-D

300 x 7.5 mm

Eluent Toluene

Flow rate 1.0 ml/min

Column pressure falls as

temperature increases due to

reduced viscosity

1260 Infinity Thermal Column Compartment

The main features are:

• Heating from ambient up to 80 °C with high heating

speeds for maximum application flexibility and

stability.

– Temperature precision of ± 0.15 °C

• Holds up to three 30 cm columns and optimized

design gives minimum dead volumes and maximum

efficiency.


22

The Agilent 1260 Infinity Thermostated Column Compartment is a stackable

temperature-controlled column compartment for LC. It is used for heating to

meet extreme requirements of retention time reproducibility.

1260/1290 Infinity TCC Options


23

1260 / 1290 Infinity TCC

G1316A 1260 Infinity TCC

G1316C 1290 Infinity TCC 100

°C

80 °C

Ideal for most applications:

Tetrahydrofuran (THF)

Chloroform

Buffers

Dimethylformamide (DMF)

Dimethyl sulfoxide (DMSO)

Toluene

Available to extend applications

up to 100 °C including Peltier cooling

to 10 o C.

Excellent Accuracy

New level of molecular weight data precision


24

The Agilent 1260 Infinity

Series column oven offers

high precision temperature

control to ensure the most

accurate results.

From 10 degrees below

ambient to 80 °C

Differential Refractive Index Detector

26

The most commonly used detector in GPC, "Universal" detector

• Monitors difference in refractive index of eluent stream as solutes emerge

from column with respect to a static reference cell filled with the pure

solvent

• Can give positive and negative peaks

• Must have sizeable difference in refractive index of solvent and solutes

• Extremely sensitive to pressure and temperature fluctuations

• Modest sensitivity, unsuitable for low solute concentrations

• Non-destructive to sample

• Easy to use

• Approximately linear response with concentration

Baseline Drift with Refractive Index Detection

27

May be caused by the column settling down

• Make sure you allow sufficient time for column to equilibrate

Can be caused by the detector equilibrating

• Allow time to reach stability - very common for RI detectors

• Ensure detector is not in a draft or direct sunlight

Baseline variations can also be cause by RI Reference cell

contents decaying or degrading, especially at temperature

• Purge the reference cell with fresh mobile phase

1260 Infinity Refractive Index Detector

Features & Benefits

Universal detector for GPC/SEC

Short setup and warm-up time

Excellent baseline stability for

GPC/SEC performance

High sensitivity, outstanding signal to

noise

Thermostated for optimal stability

Solvent recycle valve for low cost of

ownership


28

UV Detectors

29

• Relies on UV absorbing groups being present in solute

• Very sensitive detector with small cell volumes and therefore low system

dispersion

• Good linearity

• Insensitive to temperature and pressure fluctuations

• Many polymers do not have chromophores

• Many solvents or solvent additives absorb UV and either prevent use or cause

decrease in sensitivity.

• Sometimes used in conjunction with RI for copolymer analysis when only one

of the monomers has UV chromophore.

1260 Infinity Variable Wavelength Detector

Features & Benefits


30

Features Benefits

New electronics and optics offers lowest

detector noise (<± 1.5 µAU) and lowest

baseline drift (< 1 x 10-4 AU/h)

For precise quantification of trace levels

(most sensitive VWD)

80 Hz data rate Up to 100% resolution gain in fast LC

Electronic temperature control (ETC)

Maximum baseline stability and practical

sensitivity under fluctuating ambient

temperature and humidity conditions

Linear range up to 2.5 AU

For reliable, simultaneous quantification

of primary compounds, by-products and

impurities

Built-in holmium oxide filter Automatic wavelength verification

Interpreting Chromatograms

31

• The data obtained in a GPC experiment will be in the form of a chromatogram

showing detector response as a function of retention time

• There are fundamental parameters that are present on all chromatograms

Cost-effective, routine polymer characterization with refractive index and UV-visible based

on the reliable Agilent 1200 Infinity Series LC modules.

Agilent 1260 Infinity Dedicated GPC System


32

Performance and Reliability

Excellent flow rate precision

Precise retention times for reproducible, accurate molecular weights.

Extreme Robustness

Proven robust design for long instrument uptime.

Reduced operating and maintenance costs mean

a low total cost of ownership

New Agilent GPC/SEC Software

• Controls the system

• Collects the data

• Processes the results

1260 Infinity Multi-GPC/SEC System

Accurate, high performance GPC

Absolute GPC/SEC Results

The system is based on the precise and

reliable Agilent 1260 Infinity LC modules,

enhanced with the 1260 Infinity GPC/SEC

Multi-Detector Suite, the 1260 MDS.

This addition allows any combination of up to

three detector modules, in series, making this

the premium solution for GPC.

The detector modules available are:

• Dual Angle Light Scattering Detector

• Viscometer

• Refractive Index Detector

http://www.chem.agilent.com/Library/datasheets/Public/5990-9921EN.pdf

Smallest volume, highest performance

Setting the industry standard in flow cell technology

Ultra low dispersion – Ultimate in accurate, reproducible molecular weight.

Building on the UHPLC capabilities of the Agilent 1260 Infinity Series, the 1260 Infinity MDS is minimizing dispersion in GPC. The dual angle light scattering flow cell has a market-leading cell volume of just 10 μL. The high back-pressure RI cell is just 6 μL.

The viscometer is based on the latest advances in silica technology to create the ultimate balanced bridge.

Excellent baseline stability

Each detector can be individually heated to 60 °C to ensure stable baselines and excellent signal-to noise performance. Advanced optical design drastically reduces baseline spiking in Light scattering detector traces commonly seen at low angles.

Comprehensively better!

Increase performance

for your polymer analysis

richard lentner comprehensively better! applications ... · effect of injector loop size on...

Documents