Beginner's Guide to Size-Exclusion Chromatography

Which columns should I use and why?

Column selection is critical to ensure that one obtains the correct molecular weight distribution of the polymer of interest.

Initial factors to consider: Solubility

Water soluble:

• Ultrahydrogel column line

Organic soluble:

• Styragel HR for high resolution work
• Styragel HT for high temperature work
• Styragel HMW for ultra high molecular weight polymers

What solvent should I buy my columns packed in and why?

HR, HT, and HMW columns are packed in either:

• THF
• Toluene
• DMF

Specialty columns packed in methanol specifically for analysis at room temperature with HFIP (hexafluoroisopropanol) are available. If you are using a solvent other than these four for your application, there are a couple of rules of thumb to think about. If you are doing a "room temperature" application in a solvent such as chloroform or methylene chloride, convert over from THF. If you plan on doing high temperature work in TCB, ODCB, for example, convert over from toluene at ~85–90 °C.  If you are going to use a solvent that is very polar, such as DMAC (Dimethylacetamide) or NMP (n-methylpyrollidone), convert over from DMF.

I currently have columns in solvent "A" and wish to change to solvent "B"?

Generally, one can switch directly from one solvent to another at 0.1–0.2 mL/minute (see your column care and use manual) if the two solvents are miscible. If the solvents are not miscible, an intermediate solvent (which both solvents are miscible in) will have to be used.

In which order should I place the columns and why?

Generally, it does not matter what order the columns are placed in. The order will not affect the molecular weight distribution calculations of the eluting polymer. It is a good idea, however, to always place the 100 Å or 50 Å columns at the end of the set, as the styrene/divinylbenzene gel in these columns tend to be softer and less durable.

What flow rate should I use in my GPC column?

It is recommended not to exceed 1.0 mL/min for the 7.8 mm i.d. analytical columns. The "optimum" resolution for these columns is approximately 0.70 to 0.80 mL/min. The optimum flow rate for the 4.6 mm i.d. narrow bore columns is 0.3 to 0.35 mL/min. See your care and use manual for more details.

Should I gradually increase flow and temperature when starting up columns?

It is mandatory to ramp up the flow rate for analytical GPC columns, particularly the HR series. Sudden increase in flow (and subsequently pressure) will certainly damage the columns. Temperature ramping is not as critical. Generally, we ramp the flowrate from 0.0 to 1.0 mL/min over a 60 second interval, and the temperature from ambient to 150 °C (as and example) over several hours.

How do I choose the pore size range of my columns?

The range of pore sizes is chosen by determining the approximate molecular weight range of the sample of interest. If one knows that the molecular weight range is low for example, (such as an epoxy resin), than a column set of 103, 500, 100, and perhaps a 50 Å column would be used. If a medium molecular weight PVC is the sample of interest, then a 103, 104, and 105 set would be used. Choosing individual pore sizes targeted at the molecular weight range of the polymer provides the highest resolution. If the molecular weight range is not known, or is very broad, it is a good idea to use mixed bed, (i.e. "linear', or "extended range") columns which provide a mixture of pore sizes.

What is resolution and how much do I need?

In GPC analysis, resolution means range of molecular weight separated in an incremental volume of elution. We would like to maximize this whenever possible. The easiest way to maximize this is to add more columns (and therefore analysis time, unfortunately). Another way is to use smaller particle size, (~ 5 μm) which will increase efficiency. The trade-off here is column durability and lifetime. In separations where oligomers, additives, multi-modal distributions, are present, resolution may be important. If the sample is a high density polyethylene with a broad distribution, resolution may not be as important.

Waters manufactures columns in the high resolution range (HR series) which are 5 μm, the HT series, ~10 μm(good for high temperature work and multiple solvent changeovers), and the HMW series, which have 20 μm particles. These are good for very high molecular weight samples where shearing is a problem and resolution is not as critical.

What is a "narrow" standard? What is a "broad" standard?

• "Narrow" standards are those where the polydispersity is less than ~1.10. The polydispersity is defined as the ratio of the weight average molecular weight, (Mw) to the number average molecular weight (Mn).
• "Broad" standards have polydispersities greater than 1.10 and are usually the same polymer as the sample to be analyzed.

If I use "narrow" standards can I inject more than one standard at a time?

In conventional GPC with RI detection it is certainly acceptable to inject a mixture of standards, as long as there is sufficient resolution among the eluted standards. We would suggest a maximum of three. With advanced detection such as viscometry, where the area under the curve for the standard needs to be known accurately, one standard at a time should be injected.

What standard(s) should I use for my polymer?

For most people, a narrow standard "relative" calibration is fine. In this case, polystyrene standards are the usual choice for organic GPC, but PMMA's, polyisoprenes, polybutadienes, and polyTHF narrow standards may be used. For aqueous GPC, narrow polyethylene oxides, polyethylene glycols, and pullulans (polysaccharides) are available. If the user needs the "true" molecular weight (relative to the calibrant not being good enough), the broad standard (or reference) with the same chemical nature as the samples may be used.

How reliable are broad standards?

In most cases, broad standards that are commercially available have been well characterized by techniques that provide a reasonable Mw, Mn, Mz, etc. There is a certain amount of trust in purchasing these standards that the values reported are accurate and obtained with excellent precision. After all, your calibration curve is based on these values. One can also send out a representative sample that is typical of samples run in the laboratory for analysis by these ancillary techniques. Many contract labs and universities can perform these analyses and provide you with Mn, Mw, Mz, etc. on your sample that you wish to use as a broad standard.

Can I use k and alpha values from the Polymer Handbook?

The concept of Universal Calibration, developing a calibration based on log hydrodynamic volume instead of log molecular weight allows one to obtain "absolute" molecular weights for unknowns. A plot of log [intrinsic viscosity] vs. log [molecular weight] results in what is referred to as the "Mark-Houwink" or "viscosity law" plot. The slope of this curve is alpha, and the intercept determines k, (the Mark-Houwink constants). In the absence of an in-line viscometer detector, the Mark-Houwink constants may be employed, provided that they are well known for not only the narrow standards to develop the universal calibration, but also the unknown. The values found in the Polymer Handbook must be for the correct polymer of interest, in the correct molecular weight range, in the solvent being used and at the temperature of operation.

How do I prepare my mobile phase?

In most cases, the only step required to prepare the mobile phase is filtration. The solvent should be filtered through a 0.45 μm (micron) fluorocarbon filter, (acetate type for aqueous GPC).

What additives are important and when should I use them?

In certain cases, some mobile phase additive is required. For example, 0.05M Lithium Bromide is added to polar solvents such as DMF, DMAC, and NMP. These polar solvents are used to analyze polar polymers such as polyurethanes or polyimides, and there is a dipole interaction that occurs, causing artificial shoulders to appear on the high molecular weight end of the distribution. This interaction is eliminated with the addition of the salt. In high temperature analysis of polyolefins, approximately one gram per 4 liters of an antioxidant (most any hindered phenol will do) needs to be added to the mobile phase (TCB, for example). This will help to reduce oxidation of the sample as it sits in the sample carousel at high temperature prior to injection.

How do I prepare my samples? (i.e. temperature, time, and mixing)

The main question one needs to ask before trying to do GPC analysis is: What is my sample soluble in? Waters began as a company doing GPC, and we have developed a long list of solvents and temperatures for almost every polymer that has ever been run by GPC. The amount of time the sample takes to dissolve (whether room temperature or elevated temperature) usually depends on two things: the molecular weight and the crystallinity. The higher the molecular weight and the more crystalline the polymer, the longer it takes to dissolve. Usually, two to three hours with slight stirring will dissolve the sample. In some cases, (for example, ultra high molecular weight polyethylene, for example) several hours are required. High speed mixing, ultrasonic dissolution, and microwave dissolution should be avoided, unless carried out without any degradation to the polymer.

What concentration and injection volume should I use?

As a rule of thumb, a polymer with a peak molecular weight of 100,000 should be prepared in the solvent at a concentration of ~0.10–0.12%, (weight/volume). This represents approximately 1 to 1.2 mg. of sample (or standard) per ml. of solvent. As you go up in molecular weight, the concentration should decrease accordingly. A high molecular weight polymer (such as ~3,000,000 weight average) should be analyzed at a concentration of <0.02% (w/v). On the other hand, an epoxy resin with a molecular weight under 1,000 can be run at a concentration of 0.20%.

At these concentrations, the maximum injection volume per 7.8 x 30 mm column should not exceed 100 μL.

What advantages does having a viscometer and/or light scattering detector on-line give me?

As polymer characterization chemists wish to obtain more information about their specific samples, more people are leaning towards "advanced detection" techniques. Having a viscometer on-line with the refractive index detector gives you three main advantages over having just an RI alone:

• "Absolute" molecular weight via universal calibration
• Intrinsic viscosity of the polymer across the distribution
• Branching information as related to long chain branching

The light scattering detector will allow you to obtain:

"Absolute" weight average molecular weight (Mw) without establishing a calibration curve radius of gyration of the polymer branching information as for the viscometer.

In this Primer

Beginner's Guide to Size-Exclusion Chromatography

GPC Basic Chemistry

Calibration of the GPC System

Frequently Asked GPC & SEC Questions

Applications for Room Temperature GPC

References