Introduction to Size Exclusion Chromatography (SEC HPLC)

Size Exclusion Chromatography. Often known as “SEC”.

Other names used to describe SEC:

• Gel Filtration Chromatography or “GFC” is a commonly used phrase when you are separating biological molecules in aqueous (or sometimes organic containing mobile phases). It is often described as a gentle form of chromatography leaving the protein or sample intact (*Proteins are one of the most common molecules separated using this technique, but if needed intact, must be kept away from denaturing agents). 
• Gel Permeation Chromatography or "GPC" usually refers to the separation of polymers using an organic solvent, but water soluble polymers are also applicable too.

Basic Principle: Used to separate molecules based on their molecular size in solution (as the primary mode of separation). The pore size and interstitial volume of a packed column must be determined to find out which molecules it excludes. Small molecules which are smaller than the pore size will enter the particles and spend more time navigating the channels within than larger molecules which will be excluded from entering the particles and exit quickly. It is extremely important to measure this so you know what the actual column volume is AND what the exclusion limit is. Manufacturer’s often report these exclusion limits via calibration tables for linear standards such as dextran or polystyrene though some provide data using globular standards which provides more accurate data when running many proteins or peptides. Please keep in mind that the actual confirmation (hydrodynamic volume) of the compound in the mobile phase may be different than what any of these standards are so the best column to choose may be one with a different pore size than suggested (this is why it is so important to test your compound on actual columns). Determine the actual exclusion volume running actual samples. They should elute at the Tzero point (column void volume).

Support Types: Available supports are most commonly based on either silica gel or polymeric materials (e.g. DVB). Their properties and chemical compatibility may vary so be sure to document which back-pressure ranges, pH, flow rates, temperature and/or solvents are safe to use with them.

Technique: Improved resolution often results from chaining columns together, in-series, with the same pore size. Additionally, a broader range (size) of molecules can often be separated using multiple columns with differing pore volumes together, in-series (very common in GPC applications). Single "Mixed Pore" columns are also available from many manufacturers which allow a wide range of molecular weights to be screened, though often at reduced resolution. It is important to make sure that there is no interaction between the stationary phase used and the solute employed to transport the sample. This will insure that the only mechanism being used is size exclusion.

Misc. Method Development Notes: 

(1) As the primary mode of chromatography is based on "size", achieving acceptable K prime values for retention are not applicable in this mode. K prime is NOT applicable to ion exchange or SEC modes. You must achieve retention past the initial pore exclusion point to demonstrate that the compound(s) are interacting with the pores of the phase. Measure the actual column volume to determine Tzero (this is very important). Inject an unretained compound to confirm and record the pore exclusion limit with a suitable high Mw standard.

(2) For silica based supports, strong salt buffers are often employed. You must insure proper miscibility of the sample and mobile phase at all times. Be sure and flush the system of all buffers at the end of each day. This is critical and not an optional step if you want to maintain the chromatography hardware. Salt crystals can be corrosive to the steel used in these system and may result in damage to the pump, injector and other components if not flushed out. Use a flushing solution that is similar to your mobile phase, but without the buffer. If you see any salt crystals forming on the instrument, then you have not been flushing the system down properly, or often enough. Salt should never be visible on the outside of the instrument. 

(3) Method development using buffered mobile phase solutions may employ several key variables to achieve good results. After selecting the correct column(s) use a linear flow rate and systematically adjust: (a) the molarity of the buffer salt used (e.g. 10 mM, 50 mM, 100 mM, 0.5M ...); (b) the pH of the solution (acidic, neutral, basic); (c) the temperature of the column to achieve satisfactory resolution. Note: Selecting the best column is the single most important aspect of success. If you select a column that is poorly suited to the separation, a great deal of time and money will be spent on the method development with poor results. Start with the most suitable column(s).

pH Measurement of HPLC Mobile Phase Solutions and Buffers

Several times each month I am asked how to "correctly check and adjust the pH of an HPLC buffer solution which has an organic solvent component"? Well, the answer is to always check and adjust the pH of the purely aqueous solution first. Only pure aqueous solutions can be correctly adjusted for pH in the laboratory. Do not mix any organic solvent into the water based solution until after you have correctly adjusted the pH. The addition of an organic solution will throw off the final reading. Once the aqueous portion of your solution has been correctly adjusted to the desired pH value, then you can mix the solutions (or run an organic solvent gradient against the aqueous portion) as needed.

*This procedure also serves to make sure that all solutions used in chromatography are prepared in the same manner. It is true that the pH of the final mobile phase mixture (aqueous and organic mixture) may not be the same anymore, but the prepared stock solutions from which they were made will be the same each time, insuring reproducible results. Developing and describing chromatography methods and procedures which are highly reproducible equates to good scientific technique.

Common HPLC Calculations:

Capacity Factor / Retention Factor / Capacity Ratio:  k1 (K Prime)

k1 = T(R) - T(0) / T(0)
where T(R) equals the retention time of the peak in minutes and T(0) is
the retention time of an unretained peak. *For chromatography to take place, K Prime must be > 1.00 and for most modes of chromatography, should be greater than 1.5 or 2.0 for all samples !


Tailing Factor: USP: 't'

t = W(5.0)/tw/2

where tw equals the distance between peak front and T(R) at 5% of peak height units. W(5.0) equals peak width at 5% height, in minutes.


Theoretical Plates: USP and ASTM, 'N'

N = 5.54 x (T(R)/W(50))2          

Assumes width at peak half height (50)
* More info can be found at this link.


Resolution: USP, ASTM, 'R'

R = (T(R)(b)-T(R)(a)) x 2.35/(W(50)(b) + W(50)(a))/2

Assumes width at half height (50%) with peaks (a) and (b).

*Notes: Visually, "Baseline" resolution is R = 1.5. Your goal should be R = or > 2.0. ** R of 1.5 provides 99.8% separation which means you cannot accurately quantify a 0.1% impurity so develop the method to have a resolution value of at least 2.0.


Note: The appropriate formula(s) for use with your samples may depend on which of the many pharmaceutical guidelines and regulations apply in your country. Always consult the appropriate guidelines.

Determining the Data Acquisition Rate (Sampling Rate) For Your HPLC Detector

Another common question I am asked is how to set-up the HPLC detector’s sampling rate. This article is specific to commonly used UV/VIS, not mass selective detectors (Mass Spectrometer detectors are set-up in a similar manner, but you also want to take into account the numbers of MRM transitions for each peak and dwell time to account for the scanning delay. Typical values for MS are >10 points with 15-20 being best). 

Most HPLC (UHPLC) instrument manufacturer’s provide default sampling rate values within their software packages. Please do not use them as the values shown were just put there to fill in the data field and may not apply to your application or method. Many chromatographer's use these values without first understanding if they are appropriate for their own methods. This is a common mistake. Just as the manufacturer does not know what wavelength, flow rate or mobile phase you will use, they also do not know what sample(s), method and/or conditions are appropriate for your specific application. As such, they provide numerous default values in these data entry fields to satisfy the software's requirement. Just as you select an appropriate wavelength and bandwidth, you should always calculate and enter the correct detector data acquisition rate value yourself which is appropriate for your specific application, detector type and method. 

The Peak shape's role during integration: For each chromatographic analysis you must determine the optimum sampling rate for the chosen detector. An accurate value is critical for proper instrument set-up, quantification and integration of your sample(s) peaks. In the most basic sense, the area under a perfectly Gaussian peak requires at least ten points to describe it with some detail. Ten points will provide basic data about the shape of an ideal peak to the computer. Since peaks are rarely perfectly symmetrical, a larger number of points will provide more accurate integration of the peak’s actual shape and total area. This will improve run-to-run reproducibility and quantification. We suggest you include twenty to thirty data points to allow for a more detailed fit to the peak. Too few points across a peak and you lose detail and sacrifice reproducibility. Too many points and you start to introduce noise into the system. 


With these facts in mind we can next think about calculating the detector’s data acquisition rate. You must select a data rate (sampling rate) that is sure to provide the recommended 20 to 30 data points across the peak width (we use the commonly calculated peak width at half height as the time measurement). Select a detector sampling rate that will provide you with this degree of detail and resolution. This is best accomplished by initially looking at an actual chromatogram of your sample. Look at the chromatogram and use the narrowest sample or standard peak past the void time, with good retention as an example to determine the best acquisition rate. The narrowest peak will be the worst-case scenario and will insure that you have enough points across all of the remaining peaks in the sample. It's width is often measured in units of time (seconds/minutes). This data can often be read directly off of a generated data acquisition report.

Examples:

(a) If your narrowest peak has a peak width of 1.00 minute (60 seconds), then divide 30 points into 60 seconds for a result of 2 seconds per data point. The preferred sampling rate would be 2 seconds, 0.03 minutes or 0.5 Hz (depending on the units used by your detector).

(b) If your narrowest peak has a peak width of 0.20 minutes (12 seconds), then divide 30 points into 12 seconds  for a result of 0.4 seconds per data point. This equals a sampling rate of 2.5 samples per second or 2.5 Hz.

Summary:  

     To Determine the Data Acquisition Rate For Your Detector You Need To:

• Calculate the best data rate for each method and not use a generalized value (though similar methods will often use the same rate).
• Use your existing sample integration data results to identify the narrowest chromatographic peak in your analysis (at the baseline or half-height).
• Record the width value of this peak (usually in units of time).
• Divide this number by thirty (30) to determine the preferred sampling rate.
• Use this value, or a value close to it, for your detector’s sampling rate.