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)”

Chromophore, Chromophores, UV Absorbing for HPLC Analysis and Detection

A compound's absorption coefficient relates to its "strength". I find it useful to know which compounds can (and cannot) be easily detected by UV/VIS and a quick analysis of their chemical groups can provide an answer. Please note that the actual measured absorbance maximums will vary depending on the solution that the compound is dissolved in. Beta-Carotene is included as a very interesting structural example because it is composed of long chains of conjugated double bonds (isoprene units) which are cyclised at each end. Here are some other popular examples:

KEY CHROMOPHORE        Absorption MAX (nm)  STRENGTH

acetylide                                    177                           medium
aldehyde (2)                                210                           strong
anthracene                                 252 & 375                strong
azido                                          190                           medium
amine                                         195                           weak
benzene                                     184 & 255                strong
β-carotene                                  450                          medium
disulfide                                      194                          medium
ether                                           185                           weak
ethylene                                     190                           medium
ketone (2)                                   190                           weak
naphthalane                              220 & 286                strong
nitrate                                        270                           weak-strong
nitrite                                         225                           weak
nitro                                           210                           strong
oxime                                         190                           medium
thiol                                            195                           weak
thioketone                                  205                           strong
thioether                                     194                           medium
conjugated ring                        varies                          strong

Notes: 

1. Chromophore conjugation is the process that gives rise to multiple spectral peaks (or shoulders) which are very useful in qualitative identification for HPLC (Spectral fingerprinting). For more information on this topic, I recommend a very well written description of UV/VIS spectroscopy fundamentals at this link.
2. Other interesting examples: Carbonyl (aldehyde) as found in Acetaldehyde; 293nm. Carbonyl (ketone) such as found in Acetone; 271nm.

Data supplied from "Instrumental Methods of Analysis"; Willard, Merritt & Dean; D. Van Nostrand Co. Inc., (1965).