Method Development | HPLC Q&A | Technical Notes & Support | HPLC

Method Development

	Q1	How much solvent should be flowed until it becomes stable?
	Q2	How do I adjust retention using an organic solvent for analysis in reversed phase mode?
	Q3	What is the salting-out method?
	Q4	What precautions apply to the ion-pair method?
	Q5	If salts are added to a mobile phase in reversed phase mode, the retention increases. Does this phenomenon occur for both neutral compound and ionic compounds?
	Q6	How do I adjust retention using an organic solvent for analysis in normal-phase mode?
	Q7	How do I adjust retention using an organic solvent for analysis in ion exchange mode?
	Q8	What precautions are necessary when setting the conditions of an ion exchange column?
	Q9	What should we note about analysis with a buffer-type mobile phase?
	Q10	How should the solvent be replaced when changing a CN or NH2 column from the reversed phase to the normal phase?
	Q11	How should the conditions for changing a conventional column to a semi-microcolumn be set?
	Q12	Can the CAPCELL PAK be used for analysis under the same conditions as a conventional ODS column?
	Q13	What is the usual pressure for analysis?
	Q14	What is the allowable temperature of a column?
	Q15	How should the internal standard compound be selected?
	Q16	How should I prepare a basic mobile phase whose base or salt can be removed after collection of fractions?

Q1	How much solvent should be flowed until it becomes stable?

For analysis with a simple mobile phase like aqueous or methanol liquid in reversed phase mode, keep the liquid flowing for about 30 minutes. For analysis with an ion-pair agent or buffer solution, keep the liquid flowing for 5 hours or longer or preferably overnight. Note that the stabilizing time differs depending on the detector. For carbohydrate analysis with aqueous or acetonitrile liquid using NH2, keep the liquid flowing for 4 to 5 hours. For analysis with a mobile phase containing buffer solution (use in ion exchange mode), keep the liquid flow overnight. In general, ion exchange modes require longer conditioning time than other modes.

Q2	How do I adjust retention using an organic solvent for analysis in reversed phase mode?

In reversed phase mode, a mixture of water solution and organic solvent is used as a mobile phase. Increasing the organic solvent ratio decreases the retention, while increasing the water ratio increases the retention. The elution strength differs with the organic solvent type. Since acetonitrile has greater elution strength than methanol, for example, its retention is smaller for the same composition. (Reference: P.J. Schoenmakers et al., J. Chromatogr. 218, 261(1981))

Q3	What is the salting-out method?

The salting-out method is a technique of controlling the retention of highly ionic compounds in reversed phase mode. Since sulfonic acid and quarternary ammonium do not allow ion suppression by pH adjustment, adding salts increases retention in reversed phase mode. This is called the salting-out method. For the salting-out method, sodium perchlorate that is highly soluble in an organic solvent is often used and the appropriate concentration is 100 to 200 mmol/L.

Q4	What precautions apply to the ion-pair method?

1. Add about 5 to 10 mmol/L of the ion-pair agent.
2. Set the pH of the mobile phase to a value (pKa±2) where the sample is adequately ionized.
3. Dissolve the sample in the mobile phase.
4. Allow enough equilibrating time.
5. Do not inject a large amount of sample at a time.
6. Note that the calibration curve is not linear in a wide range.
7. Do not use a column that has been used by the ion-pair method for different purposes, because the ion-pair agent may remain on the surface.

Q5	If salts are added to a mobile phase in reversed phase mode, the retention increases. Does this phenomenon occur for both neutral compound and ionic compounds?

The influence of salts in a mobile phase on the solute retention can be explained by the solvophobic theory. In the model of solvophobic theory, hydrophobic molecules are assumed to be ousted from a polar solvent to a hydrocarbon-bound phase. According to this theory, hydrophobic interaction is supposed to be generated from the repulsive forth between a polar solvent and a non-polar solute and stationary phase solid. If a neutral inorganic salt (e.g. potassium chloride) is added to the mobile phase, the electrostatic repulsion between solute molecules decreases and the surface tension of the eluent increases. Therefore, the retention increases linearly with the salt concentration. The influence of added salt on the retention of ionic compound has a more complicated mechanism than that on a neutral compound. Irrespective of the compound, however, salt addition increases the retention. For the analysis of a compound with a sulfonic acid moiety with a reversed phase column, add an ion-pair agent or inorganic salt to increase the retention. For example, sodium alkylsulfate, a surfactant, can be analyzed by adding potassium chloride of about 0.5 mol/L to a mobile phase. To protect the whole system from salt precipitation, the maximum amount of salt addition is 1.0 mol/L and enough retention and separation are often available at about 0.5 mol/L. If the sample is hydrophobic and the mobile phase contains a large amount of organic solvent (e.g. alkyl sulfonic acid sodium of 10 or longer chain), sodium perchlorate that dissolves well in an organic solvent may be used. Sodium perchlorate is expected not only to increase the retention by increasing the surface tension of the mobile phase but also to neutralize the positive charge of a basic compound by perchloric acid ions as a hydrophilic ion-pair agent. Therefore, sodium perchlorate is an effective additive for separating a quarternary ammonium. (Reference: "Reversed phase High Performance Liquid Chromatography" by Tokyo Kagaku Dojin).

Q6	How do I adjust retention using an organic solvent for analysis in normal-phase mode?

A6	In normal-phase mode, a mixture of two or more organic solvents (e.g. n-hexane and ethanol) is used as a mobile phase. Raising the ratio of a solvent of greater polarity reduces the retention. On the contrary, raising the ratio of a more hydrophobic solvent increases the retention.

Q7	How do I adjust retention using an organic solvent for analysis in ion exchange mode?

In ion exchange mode, separation is achieved by hydrophobic interaction as well as ion exchange. To adjust the retention in ion exchange mode, therefore, change the organic solvent, salt concentration, and pH. The retention becomes smaller as the organic solvent concentration and the salt concentration become greater.

Q8	What precautions are necessary when setting the conditions of an ion exchange column?

Note the following three points:
1. Do not let 100% water through.
2. Take great care about salt precipitation because an aqueous solution containing salts is used for the mobile phase together with an organic solvent.
3. Control pH precisely. The suitable solvent differs depending on the compound to be analyzed. Any concentration of organic solvent (except 100% water) does not pose a problem to the packing materials but take great care about salt precipitation in a column. Make sure that no precipitation will occur in the column, not only when preparing a mobile phase but also replacing it.

Q9	What should we note about analysis with a buffer-type mobile phase?

1. When letting a buffer-type mobile phase through the column, prevent salt precipitation by fully considering the mobile phase remaining in the column. If the column has a mobile phase containing a lot of organic solvent, salt precipitation may occur. Replace the mobile phase with a low organic content solution or organic solvent of the same organic composition as a buffer-type mobile phase, and then change it to the mobile phase you are going to use.
2. When storing a column for several months, replace the mobile phase with one having all the components but salts. For short-term storage, the column life will be longer without rinsing. For daily routine use, keep the liquid flow at a low rate (0.1 to 0.5 mL/min) throughout the night.
3. Allow an equilibrating time of at least 5 hours or one night before analysis.
4. When analyzing an ionic compound, set the pH of the mobile phase at the value adequately apart from the pKa of the compound (2.0 or more). Around pKa, the good reproducibility is not expected because the retention time fluctuates with slight changes of the mobile phase pH.

Q10	How should the solvent be replaced when changing a CN or NH2 column from the reversed phase to the normal phase?

A10

Take the following steps:
1. Replace with the solvent of a mobile phase minus salts.
2. Replace with methanol.
3. Replace with chloroform (or acetone).
4. Replace with the object solvent.
Let the liquid flow through thoroughly at each step. Replace the rinsing liquid of the autosampler also in the order of steps 2 to 4 above (since the autosampler uses the solution of 1 in advance, skip step 1).

Q11	How should the conditions for changing a conventional column to a semi-microcolumn be set?

A11

For a semi-microcolumn, reduce and set the flow rate in proportion to the cross section of the column. When using a semi-microcolumn smaller than 2.0 mm in inner diameter, use a system supporting semi-microcolumns (replacement with supporting parts). If the same packing materials are used for a conventional column and a semi-microcolumn, a chromatogram of almost the same retention and sensitivity improved by the concentrating effect can be obtained at the above flow rate with the above equipment. However, if a column of 4.6 mm i.d. x 250 mm established as a condition is made by another manufacturer or its brand is different from that of a semi-microcolumn, it may be necessary to adjust conditions because the separation characteristics may be different. However, there is no need to develop conditions from the beginning; a slight adjustment of the existing method is enough. If a different separation pattern must be used due to the properties of packing materials (not the semi-microcolumn size but the packing material type), consult Osaka Soda.

Q12	Can the CAPCELL PAK be used for analysis under the same conditions as a conventional ODS column?

A12	The conditions are basically the same. If the characteristics of packing materials differ from those of the current column, the organic solvent ratio in a mobile phase or pH may require fine adjustments.

Q13	What is the usual pressure for analysis?

A13	The pressure is 15 MPa or less and about 10 MPa. Note that the instruments will receive a heavy burden if the pressure exceeds 20 MPa and also that large pressure fluctuations during analysis will produce a heavy burden on the column and instruments and cause them to deteriorate.

Q14	What is the allowable temperature of a column?

A14	The allowable temperature is about 60°C when the mobile phase is acetonitrile, methanol, etc., but about 40 to 50°C for any other mobile phase. Note that a column tends to deteriorate faster if the temperature is raised under alkali or acid conditions.

Q15	How should the internal standard substance be chosen?

A15

As the internal standard substance, a compound satisfying the requirements of stability, retention close to (preferably greater than) that of the substance to be analyzed, and no existence in matrix is suitable. To make a choice, it is recommended to search application data of analysis with a mobile phase of similar composition and refer to the data of analyzed compounds.

Q16	How should I prepare a basic mobile phase whose base or salt can be removed after collection of fractions?

A16	Add an amine like triethylamine to a fully degassed mobile phase for pH adjustment. A buffer solution of ammonium carbonate and aqueous ammonia can be removed by an evaporator.

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