Viscotek Standards Selection Guide

Introduction

The Viscotek range of GPC/SEC systems and detectors includes a wide selection of calibration standards for convention and universal calibration, and for triple detection. This technical note provides an introduction to the standards as well as providing some of their technical details.

Figure1: A selection of Viscotek GPC/SEC standards

Types of standard

A range of different types of standard are available:

• Triple detection standards are used for calibration of advanced detectors including light scattering (RALS/LALS/MALS) and viscometers. Two standards are provided. The first is a calibration standard used to characterize the response factors of the detectors. This typically has a narrow molecular weight distribution and peak. The second is a verification standard used to test whether the calibration has been successful. This typically has a broader molecular weight distribution. An instrument is considered to be correctly calibrated if the molecular weight and intrinsic viscosity are measured to within 10% of the certified values. It is recommended that the two standards should always be run together to ensure that the instrument is correctly calibrated.
  Malvern's triple detection standards are pre-weighed into vials so that the only sample preparation required is the addition of the mobile phase of choice. Typically, 10 mL of mobile phase is required. For SEC-MALS 20, detector normalization can be improved if the standards are dissolved in 5 mL to achieve a slightly higher concentration.

• Conventional/Universal calibration standards are used for column calibration. Each standard is run in turn and the retention volumes of each are used to build a calibration curve of molecular weight as a function of elution volume. These standards usually have a narrow molecular weight distribution. Malvern offers standards that are pre-weighed into vials so that the only sample preparation required is the addition of the mobile phase of choice. Typically, 10 mL of mobile phase is required. 

• Bulk standards are larger amounts of polymer which must be weighed out and dissolved to an appropriate concentration before use. These are available for conventional/universal calibration only.

Standards for synthetic polymer applications

Polystyrene is the most commonly used standard for organic applications and is used in solvents such as THF, toluene, TCB and ODCB. PMMA is mostly used in polar organic solvents less appropriate for polystyrene such as DMF, chloroform and acetone, but can also be used in THF, DCM, DMAc, NMP, and HFIP. Pullulan can also be used for DMSO (for details, see the section on aqueous standards).

Triple detection standards

Malvern offers a large selection of triple detection standards for organic applications. Polymer types available are polystyrene (PS) or PMMA.

Conventional/Universal calibration standards

Malvern offers a set of pre-weighed polystyrene conventional/universal calibration standards.

Bulk standards

Malvern offers a selection of polymer standards for manual weighing, dissolution and conventional/universal calibration.

Standards for aqueous applications

PEO is the most commonly used aqueous standard for calibration.  Pullulan is also commonly used, particularly in applications in acetic acid (and also for DMSO).  Dextran is commonly used for verification.

Triple detection standards

Malvern offers a range of triple detection standards for use in aqueous applications.  These include pullulan and PEO for calibration and pullulan and dextran for verification.

A separate set is offered for calibration of the SEC-MALS 20.  This has a slightly higher molecular weight calibration standard to improve the normalization of the different detectors.  The verification standard is also larger to allow for a test of the Rg measurement; however, the Rg value is not certified.

Conventional/Universal calibration standards

Malvern offers a set of pre-weighed PEO conventional/universal calibration standards.

Bulk standards

Malvern offers a selection of pullulan standards for manual weighing, dissolution and column calibration.

Standards supplied with systems

When shipping a new system, Malvern usually includes a selection of triple detection standards.  These kits include a mix of standards for organic and aqueous applications.  Only those that are appropriate for the application (organic or aqueous) should be used.  These mixed kits are not available for sale but the standards that are included are available in their own distinct kits which can be found in the previous sections.

Calibration for protein measurements

Unfortunately, Malvern is unable to offer triple detection standards for protein measurements.  We recommend that you use a fresh sample of bovine serum albumin (BSA) and calibrate using the monomer peak whose molecular weight is well established.  A procedure for doing this is available from the website.

What is an appropriate molecular weight for the standard?

For triple detection standards, the calibration standard must be small enough to be an isotropic scatterer to allow correct calibration and normalization of light scattering detectors.  For a MALS detector, a small amount of anisotropic scattering is acceptable to aid detector normalization.  For polystyrene the upper limit is approximately 110 kDa.  For PEO the upper limit is approximately 70 kDa.  For pullulan it is approximately 120 kDa. 

A triple detection calibration is a single point characterization of the instrument response factors.  It is not necessary to use a molecular weight standard close to the sample molecular weight because the instrument response is linear over the entire measurement range.

If desired, a verification standard could be used that is close to the sample molecular weight, however this is not a requirement to show correct function of the system.  A range of options are available in the conventional/universal calibration kits but these are not available individually.

For conventional/universal calibrations, the samples being measured should be within the full range of the calibration curve.

How often is calibration required?

The frequency of calibration depends on the application but these rules can help you decide whether re-calibration is required.

Triple detection calibration

Calibration of a triple detection system is theoretically a one-time event since light source power and the sensitivity of the detectors should not change.  In practice, calibration also includes analysis of inter-detector volumes, and band broadening and tailing factors, which are all dependent on the chromatography as well.  It is therefore best practice to calibrate with every sample sequence.  If this is impractical, simply run a verification standard.  If the measurements of the verification standard are within 10% of the certified values then the calibration is probably still robust.  If the chromatography is changed significantly (for instance, if the column set, mobile phase, flow rate etc. is changed), then the system should be re-calibrated for the best results.

Conventional/universal calibration and bulk standards

Column calibrations such as conventional and universal calibration are extremely sensitive to changes in the chromatography.  Any time the chromatography conditions are changed, the calibration curve must be re-created.

Standard Traceability

All Malvern molecular weight standards are supplied with a certificate of analysis.  If one has not been received, please contact your local representative.

Malvern's pre-weighed triple detection standards and bulk standards are effectively 'transfer standards'.  These are characterized on a reference system that has been shown to be well calibrated by the measurement of a NIST standard.  For organic standards, the reference NIST standard is SRM 1478.  For aqueous standards, the reference NIST standard is SRM 1423.

The pre-weighed conventional/universal calibration standards are quoted with a weight-averaged molecular weight (Mw) that has been measured in the same way as the triple detection standards.  They are also quoted with an Mp (peak molecular weight) for the creation of the calibration curve.  These values are historical values, which are not traceable.

Standard preparation

Pre-weighed standards should be dissolved in 10 mL of the mobile phase.  Pre-weighed standards for the SEC-MALS detectors should be dissolved in 5 mL of mobile phase as the higher concentration aids with detector normalization.

Samples should be allowed to dissolve for at least 4 hours and ideally, overnight.  Slow mixing or shaking can aid dissolution but ultrasound should not be used as this can degrade the standards.

Mobile phase should be dispensed as accurately as possible using a pipette.  In the case of viscous or volatile solvents, weighing the mobile phase may improve accuracy.

Calibration standards and mobile phases

The following table can be used as a guide to suggest which standards will be most appropriate for a given mobile phase:

Standard storage

If kept dry and out of direct sunlight, the standards should be stable for up to five years.

Once prepared, standards will remain stable for up to 7 days if the vial is kept sealed and out of direct sunlight.  For longer term storage standards dissolved in organic solvents should be aliquoted and kept in the fridge.  For longer term storage, aqueous standards should be aliquoted and frozen.  Standards should not be frozen more than once.

Standard disposal

If they have not been used, the standards vials should be disposed of in the same way as glass.  If the standards have been prepared, the solution should be discarded of as waste solvent and the vials in the same way as glass.