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Q: How do I prepare my Molecular weight standards or samples for GPC Analysis?  


A: Sample Preparation for SEC

1) Prepare fresh standard solutions to assure accurate concentrations. Exact concentration values are critical when using a light scattering, viscometry or Triple Detection systems.

2) Select the appropriate clean, dissolution container (auto sampler vial, bottle, flask, etc.).

3) Add an appropriate quantity of standard using an analytical balance. Start with the concentrations provided below as guideline; select your working concentration based on detection type and number of columns in use. 


Molecular 
WeightRange

 

Concentration (g/l) or (mg/ml)

 

100 - 10,000 D

1 to 2 is best for a narrow distribution standard. 

10,000 - 100,000 D

1 to 2

100,000 – 500,000 D

1 to 2

500,000 - 1,000,000 D

1 to 2

1,000,000 D and above

0.5 or less

2.0 - 4.0 mg/ml - broad standards usually.


4) Add the correct volume of solvent via syringe or pipette. To determine sample concentration it is important to add the solvent volume accurately. 

5) Note: If you use an internal standard for flow rate monitoring, please mix it with a fraction of mobile phase in the quantity you normally use, i.e. Toluene in THF (tetrahydrofuran) or (ethylene glycol in water) mobile phase and use the marked solvent to prepare your standards. Do not add it into the general solvent bottle.

6) Close or seal the dissolution container and let sit at room temperature for 24 hours or overnight for complete dissolution.

a) Let polymers of < Mw of 200,000, sit for 3-4 hours. 

b) Dissolution time for Ultra High Molecular weight standards and samples (>2,000,000) may take from 1-3 days.

7) Gently swirl the vials for a homogeneous solution.  WARNING: Do not use stir bars, ultrasonic baths, microwave heating or harsh shaking as this can cause sample degradation.

8) Standard Mixes: It is possible to dissolve up to 3 standards of the same type in a given dissolution container.  If you make your mixture, the standards to be mixed in this way must be separated by orders of magnitude in molecular weight to prevent co-elution; i.e., Polystyrene, 500, 50,000, 500,000 D.  PSS offers pre-made mixtures or kits that prevent co-elution. See ReadyCal Kits.  Note: separation will depend on the column(s) you use.  Note: When using a viscometer, your standard mixture must reach the baseline between each standard injected in order for the standard to be used in a calibration curve. 

9) Inject each solution separately. Start with the guideline below to develop your optimum injection volume
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Number of columns
(length 30 cm)

Guideline injection volume (µl)

 

4-5 and more

200 to 250

3

100

2

50

1

20

10) Ultra High Molecular Weight Samples and Standards perform best in low concentrations, through columns of large particle and pore sizes and at reduced flow rates i.e. from 1.0 ml/min. to 0.5 ml/min. This reduces the probability of sample degradation.   Inject 100 µl of 0.1 g/l instead of 20 µl of 0.5 g/l.

11) Use Mp values to construct a calibration curve.

 

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