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Wako FAQ -Chromatography- Q1

Q1: Which reference standard do you recommend for use to check performance of a column?

  • We use 3 components, uracil, benzene and naphthalene, to check performance of a column packed with a reverse phase filler.

  • Uracil is used to check the retention volume of unretained compound (V0).

  • For checking column performance, at least 2 components (benzene and naphthalene) are required.

  • In addition, the substances shown in the table are used to check the performance of metal coordination compounds, basic compounds, acidic compounds, etc.

  • The method to use the sample to be analyzed actually as the standard is also practical provided that the substance is stable.
Table: Substance groups used for checking column performance
Metal coordination compounds Acetyl acetone, phenyl acetyl acetone, hinokitiol, 8-quinolinol
Basic compounds Aniline, N-methyl aniline, N,N-dimethyl aniline, pyridine, 2-ethyl pyridine, caffeine
Acidic compounds Phenol
Neutral compounds Benzene, toluene, naphthalene
Deterioration of a column is unavoidable however carefully it is used. It is important to check the column periodically and grasp the present state also to prevent emergent troubles during use as much as possible.

Here, we introduce the inspection method before shipment of columns as a simple method.

In case of ODS columns, we check the number of theoretical plates (N), retention capability (k’), separation factor (α) and peak symmetry (s) using 3 components, 1) uracil, 2) benzene and 3) naphthalene.

Contamination (increased pressure) in a column filter and filler on the inlet side of a column can be checked by monitoring the column pressure.

<Inspection method>

Calculate the followings referring to Fig. 1.
As measurement with a ruler may give large errors (N in particular), just use these as approximate values. Calculation of N and S becomes easy by setting the chart speed faster and taking the peak width wider.

A decrease in value suggests deterioration of the state of filling and contamination of the filler.
A decrease in value suggests leakage of ODS group.

A decrease in value suggests a change in selectivity due to contamination of the filler in addition to causes for k’ decreases.

Large variation in values from the time of purchase suggests deterioration of the state of filling, contamination of the filler and dead volume in the connection of the column, etc.

Fig.1_Basic Chart for Checking Column Performance
In case of column inspection,
1. Column temperature should be kept constant (for example; 40°C)
2. Perform the inspection with the same amount of sample concentrations and injection volumes. (for example, 5~10μL of the sample volume (Fig.1))

Q1Which reference standard do you recommend for use to check performance of a column?
Q2Is there any simple method to change the solvent concentrations in the mobile phase once prepared?
Q3How are ODS columns, Wakosil-II HG, AR and RS, used in different situations?
Q4Ion pair reagents for low wavelength are on the market. What effects and differences do they show with actual samples?
Q5We wish to fractionate by HPLC. How can we determine the optimal separating conditions?
Q6We wish to perform fractionation by the reverse phase flash chromatography. How can we determine the optimal separating conditions?
Q7Is column performance improved by narrowing the inside diameter of a column?
Q8We are examining mobile phase conditions of HPLC.We intend to examine by changing solvents.Is there any method to estimate the elution profile in advance?
Q9We are examining the conditions of a mobile phase in the HPLC. We would like to know how to regulate retention of a sample that has a readily dissociating functional group.
Q10I have used a semi-microcolumn (2φ mm) but cannot obtain improvement of sensitivity generally offered.
I wonder what is the cause as I am sure to have used the system applicable to microcolumns.
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