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)”

Notes on Cleaning bound Protein from RP HPLC columns:

First, a few comments:

• ·         Before proceeding with any column regeneration or cleaning procedures, always refer to the specific advice provided by the column manufacturer. Approved maintenance and cleaning instructions can often be found in the product guide which comes with the new column. Their guidelines supersede these!

• ·         Columns are consumable items. After a suitable amount of use, the time and materials required to regenerate them may cost more than the purchase of a replacement column. Always have a new, spare column on hand.
  

•        Do not overload the column! This is the most common reason for column fouling, flow path contamination and sample carryover issues. In most cases, injection volume should be less than 1% of the column volume (maximum).
  

• ·         Protect your detector. Before washing or cleaning the column, disconnect the column outlet line and direct the column to waste only.

•        Column Storage solutions are not the same as column wash solutions. Never store a column in buffer or ion pairing containing solutions.

For RP supports, if buffers have been used, always start by washing the column down with ultra-high purity water and some organic solvent (e.g. Water/MeOH, 95%/5%) to remove all salts. Use about 10 column volumes to flush these off. Do not wash the column with organic solvents until you have first washed it thoroughly with high-purity filtered water.

Polymeric resins (e.g. PS-DVB) from many manufacturers can effectively be cleaned using 0.1 M Sodium Hydroxide solution or a mobile phase solution containing equal parts of isopropanol (IPA) and 1 to 3 M Guanidine hydrochloride at ~ 50 °C. Optionally, some success has been reported using other solutions such as: 5M Urea (pH 7) buffer solution; 1 M NaCl (pH 7) and even mixtures containing some methylene chloride solvent. Check with the manufacturer first as column damage/plugging may result if their directions are not followed.!

For RP silica based supports (non-SEC), we often start with a series of wash solutions. In most cases, pure water or pure organic solvents such as MeOH or ACN will not remove bound protein (common novice mistakes). An acid, base or even an ion pairing reagent is often needed to clean them. Start simple and monitor.

 

For RP silica based supports (SEC), a high salt buffer solution often releases bound proteins quickly. A mobile phase containing water plus an alcohol (methanol, IPA or ethanol) may also prove effective too.  Optionally, a solution of 0.5 M guanidine hydrochloride may effectively remove bound material.

General Advice: One of the first general wash solutions to start with (especially to remove basic compounds) is a 1% Acetic acid solution in Methanol (50/50). If desired a stronger acid such as 0.1 % Trifluoroacetic acid (TFA) or 0.1 % Formic Acid can be swapped for the acetic acid (where possible, start with a weaker acid). Use a low concentration of acid to achieve a pH of ~ 2.5. This acidic wash can be followed with a neutral solution, or if needed, a later solution where IPA or ACN replaces the MeOH used (50/50).

For extreme cases where the column has been overloaded with protein, a 5 M Urea solution has been proven effective in removing bound protein from silica and polymeric supports too. A word of caution, as the resulting pH of this strong solution may be greater than or equal to pH 9. Many types of silica based RP columns can not withstand strongly basic solutions and the silica inside may dissolve (plugging the column). Start with a lower concentration wash  first. You can always increase it later. Always read the instruction sheet carefully which came with the specific HPLC column to determine if it can be used at these high pH levels. Another salt solution that has shown some promise is 1 M sodium phosphate solution, pH 7.0. Run the salt solutions for about one hour at a moderate flow rate. Follow up all washes with rinses of mixtures of water and MeOH (80/20), then 90% MeOH/Water. 

Please remember that in ALL cases, HPLC columns are consumable items with a limited lifetime. Dispose of them properly when they are damaged or contaminated and replace with a new column. Once you have a fresh clean column to work with, prevent column fouling by developing better quality methods which utilize frequent, properly developed wash methods (using a wash solution which is stronger than your analysis mobile phase), filter all samples and be sure they fully dissolve in solution (100%). *Column fouling is not normal and can be prevented with proper training.