Determine void time for HPLC method with SAX column

[stela13_77]

Any suggestions what solvent I can use to determine the void time running on a SAX column (Phenomenex SphereClone-SAX column).
The mobile phase consists of 25 mM NaH2PO4 + 10 mM Na2HPO4.
The pH is around 6.
The HPLC is equipped with a UV detector.

[Multidimensional]

Great question. Determining the Column's Void Volume is always one of the very first steps you should take before running any samples. This is one of the most basic skills a chromatographer should know how to measure in their first week of training. Typically, to find this value we will inject a compound that is not retained (unretained) on the specific column to use as a 'marker'. An unretained sample will not interact with the support and elute off the column quickly. When flow rate is considered, the column volume can be calculated. This TIME point establishes the earliest time a 'Real" peak could elute off the column and also, how well a sample is retained on the column (a ratio of retention, often called the Retention Factor or Capacity Factor)

HPLC COLUMN VOLUME: Please Read this authoritative Article for more info, "Determination of HPLC Column Void Volume / Dead Volume, Dead Time (T zero)" [https://hplctips.blogspot.com/2011/05/d ... -time.html

However, in your example (an ION EXCHANGE and/or a Size Exclusion Column) ), the COLUMN's void volume is best determined by: (1) Equilibrating the column in the aqueous mobile phase until you observe a steady, flat baseline with minimal pump ripple (<1%). (2) With the UV detector set around 230nm (10), inject a small volume of pure methanol and watch the baseline. At some point you should observe a small injector-valve induced pressure peak (a sinusoidal 'blip') on the baseline. Make sure to adjust the 'Y' scale low enough to see it. This 'blip' results from the pressure change which occurs from switching the injection valve from the "load" to "inject" positions. Use a low UV wavelength to observe this deflection on the baseline. The retention time of this peak will correspond to the Void TIme. Knowing the Void TIME will allow you to calculate the Column's Void VOLUME (e.g. flow set to 1 mL/min. If the 'blip' is observed at 3.00 minutes, then the column's void volume is 3 mLs.

IMPORTANT: K prime is of no value or use when using Ion Exchange or Size Exclusion Columns so do not use the Column Volume value to find K prime values for various peaks. This is one of the very rare cases where K prime is not relevant. To read more about K prime, and its importance in HPLC method development, please read this article, "HPLC K Prime. Also known as: Retention Factor, Capacity Factor): One of the Single Most Important HPLC Parameters of All" [https://hplctips.blogspot.com/2015/06/k ... actor.html]

[vmu]

Correct determination of the column hold-up volume (Vm, the total mobile phase volume in the column, i.e. the sum of the column interparticle volume and the sorbent intraparticle pore volume) is not a simple task. Many papers are devoted to this problem. For example:
Pure Appl. Chem., Vol. 73, No. 6, pp. 969–992, 2001.
RETENTION PARAMETERS IN CHROMATOGRAPHY (IUPAC Recommendations 2001). PART A. HOLD-UP VOLUME CONCEPT IN COLUMN CHROMATOGRAPHY
https://www.degruyterbrill.com/document ... 60969/html

The accurate value of Vm (which is not so easy to measure) is more important for scientists than for industrial chromatographers. First of all, it is necessary to clearly understand that Vm expressed as a percentage of the column tube volume (Vc = L*pi*(dc^2)/4), i.e. Vm/Vc*100:
- can never be more than 100 % (it can be as high as 85 % in size-exclusion and in silica monolithic columns, but never higher than 100 %; some chromatographers fail to check their experimental values of Vm against this obvious limit);
- usually lies in the approximate range between 50 % and 70 % for the columns filled with porous particles (core-shell and fully porous);
- is usually close to 40 % (36 % to 42 %) for the (rarely used) columns filled with nonporous particles.

The values of relative volumes in the range from 36 % to 42 % also correspond to the typical interparticle column volume (aka "external volume", Ve; do not confuse with the extra-column volume) in the columns filled with porous particles. This is the fraction of the column volume available for the analytes totally excluded (via size-exclusion and/or ion-exclusion mechanism) from the pores of the sorbent particles. This volume is used as the dead volume is SEC (where the symbol Vo or V0 is normally used for it).

Also the interparticle volume Ve is used for the calculations of the zone retention factor k'' = (Vr - Ve)/Ve in scientific papers devoted to the band broadening theories (Vr is the analyte retention volume). k'' is used to describe the analyte distribution between the moblie zone (the interparticle volume Ve wherein the eluent flows) and the stationary zone (Vsz, the whole particles volume that includes the particle material and the stagnant eluent in the intraparticle pores) in the column.
Ve + Vsz = Vc.
k'' is the equilibrium ratio of the analyte masses in the stationary zone and in the mobile zone.

The more widely known phase retention factor is k' = (Vr - Vm)/Vm. It is used to describe the analyte distribution between the two phases. The mobile phase volume is Vm. The stationary phase quantity can be defined in several ways (as the sorbent surface area, as the solid material volume, as the bonded layer volume, etc) to link k' to the correspondingly defined distribution constant.
k' is the equilibrium ratio of the analyte masses in/on the stationary phase and in the mobile phase.

In SEC, it is common to calculate the distribution constant instead of k'. With the same notation as above, this constant is given by Ksec = (Vr - Ve)/(Vm - Ve). It describes the analyte distribution between the volume of the eluent flowing in the interparticle channels (Ve) and the volume of stagnant eluent in the intraparticle pores (Vm - Ve).
Ksec is the equilibrium ratio of the analyte concentrations in the intraparticle pore volume and in the interparticle volume.
Ksec varies in the range from 0 to 1.