HPLC COLUMN TEST MIXTURE SOLUTIONS; NP & RP EXAMPLES

When selecting standards for use in testing an HPLC column OR for evaluation of an HPLC system’s performance (e.g. System Suitability, Performance Verification, Retention), in addition to selecting special high purity chemical compounds, consider using the actual sample which is specific to the method or application. Characterize the sample’s retention time (esp. K prime), peak shape and spectra (if applicable) and use the data to measure, compare and detect changes in  performance over time. This can be combined with a suitable test mixture to provide more comprehensive results.

Common HPLC Column Test Mixtures:

Running Sugars on an Amino Column? Use simple and complex sugars as standards. Example: D-Fructose; D-Glucose; Lactose; Maltose; Sucrose.

NP (prepare the test solution in a mixture of Hexane/Ethanol, as appropriate)

Diethyl phthalate; Dimethyl phthalate; Toluene; Benzene.

RP (prepare a fresh test solution in ACN/Water or Methanol/Water, as applicable)

Select 4 or 5 of these compounds for use in a mix. Uracil; Benzene, Acetophenone; Toluene; Naphthalene; N,N-Diethyl-m-toluamide; Phenol;  diethyl phthalate; diamyl phthalate; di-n-hexyl phthalate; dioctyl phthalate.

Include a Void Marker in your Test Solution:

Always measure the actual void volume of your specific HPLC column with a compound which is un-retained by your column. For RP applications which utilize at least 20% organic, Uracil or Thiourea are often used, but some inorganic salts (e.g. sodium nitrite and sodium nitrate) have also been shown to work as well. Monitor by UV detection. 

 

• You must know what the Method's void time is for every HPLC method that you run BEFORE starting to analyze any samples. This is one of the most fundamental aspects of using HPLC so make sure you understand and can show what this value is.

Reference: “Determination of HPLC Column Dead Volume/ Dead Time (T zero)”