Preparing the Mobile Phases SHIMADZU (Shimadzu Corporation).Pd

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Preparing the Mobile Phases

HPLC A Basic Knowledge of Analysis

-Differences in Preparation Method May Result in Different Chromatograms -

A water-based solvent, organic solvent, or a mixture of the two is

mainly used as the mobile phase for HPLC. A buffer solution is often

used as the aqueous solvent. LCtalk38 LAB Preparing Buffer Solutions

describes the actual preparation methods for typical buffer solutions

used with HPLC. However, it seems that many people have a vague

understanding of buffer solutions.

If the preparation method is not the same as the method described in

the material for the analysis method, differences can occur in the

mobile phase that affect the chromatograms and analysis results. Apart

from the buffer solution, many other unforeseen factors affect the

mobile phase preparation, such as the method of mixing the solvent.

Here, we use actual examples to consider the effects of the mobile

phase preparation method on the analysis results.

1) Preparing Buffer Solutions

In practice, how do we go about preparing a buffer solution that is denoted as "20 mM phosphoric acid buffer solution

(pH=2.5)"? Let's examine a few possible issues.

First, we know that it is a phosphoric acid buffer solution, but what is the counterion? If sodium ions are unambiguously

the counterion, does "20 mM" refer to the phosphoric acid concentration or the sodium phosphate concentration?

For a 20 mM phosphoric acid (sodium) buffer solution, "20 mM" could be considered to be the phosphoric acid

concentration. On the other hand, considering "20 mM" to be the sodium concentration, the buffer solution could be a

buffer solution prepared by adjusting the pH using a 20 mM aqueous solution of sodium dihydrogen phosphate.

(However, a 20 mM aqueous solution of sodium dihydrogen phosphate has a pH just under 5, so that pH adjustment

with some type of acid is required to achieve pH 2.5.)

However, an ion-pair effect due to the acid used for pH adjustment could affect the analysis results. This is potentially

dangerous, as it leads to several possible interpretations of a denoted buffer solution.

The example above leads to three potential interpretations. Fig. 1 shows how this affects the analysis results.

In the top row, "20 mM" is interpreted as the phosphoric acid concentration. The analysis results were obtained using a

solvent prepared as "20 mM phosphoric acid (sodium) buffer solution (pH 2.5)" as the mobile phase.

In the middle and bottom rows, "20 mM" is interpreted as the sodium dihydrogen phosphate concentration. The mobile

phase was prepared by adding phosphoric acid or perchloric acid to adjust the pH to 2.5.

These differences can significantly affect the retention time, as shown for dihydrocodeine in the example, and can lead

to problems with the robustness of the analysis method. Clearly identifying the buffer solution when specifying the

preparation method can prevent problems due to differences in interpretation.

1. Acetaminophen 2. Dihydrocodeine 3. Caffeine

Analytical Conditions

Column Shim-pack VP-ODS (150 4.6 mm I.D.)

Mobile phase Buffer solution (pH 2.5) / acetonitrile = 9 / 1

Top row A 20 mM phosphoric acid (sodium) buffer solution (pH 2.5)

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Middle row

B

20 mM sodium dihydrogen phosphate buffer solution (pH2.5); phosphoric acid added

to adjust the pH

Lower row

C

20 mM sodium dihydrogen phosphate buffer solution (pH 2.5); perchloric acid added

to adjust the pH

Flowrate 1.0 mL/minute

Temperature 40 C

Detection 210 nm

Fig. 1 Effects of Buffer Solution Preparation Method

2) Mixing Organic Solvents and Water-Based Solvents

A mixed solution of organic solvents and aqueous solvents is often used as the mobile phase. The way that they are

mixed can cause large differences in analysis results. Consider an example of 90 % buffer solution and 10 %

acetonitrile. (The buffer solution is 20 mM sodium dihydrogen phosphate adjusted to pH 2.5 by the addition of

phosphoric acid.) A mixing ratio of 9:1 can be interpreted as a 9:1 volume ratio of buffer solution and acetonitrile. That

is, the equivalent volumes are measured and mixed together.

On the other hand, it is possible to interpret this as 10 % acetonitrile content, that is, that the acetonitrile must be

diluted ten times with buffer solution. This results in a reduced volume due to mixing, such that surplus buffer solution is

added. It may be felt that there is no great difference between the two. Care is required, as the type of mixing can have

a remarkable effect on the analysis results (in particular, the retention time), as shown in Fig. 2.

Generally, when preparing the mobile phase for high-performance liquid chromatography, the proportion is often

denoted as Liquid A : Liquid B = 3 : 2 (V/V). That is, the equivalent of volume ratio 3 of Liquid A and the equivalent of

volume ratio 2 of Liquid B are measured separately and mixed together. (In practice, this results in a mixture volume

slightly less than the equivalent to theoretical total "5" for the two liquids (3 + 2).)

The problem above also relates to the preparation of sample solutions and other solutions, not only to the preparation of

mobile phase.

Each field (such as pharmaceuticals and chemical engineering) has its own conventional wisdom and customs, which

causes even more confusion. Keep in mind to always use appropriate indications to avoid confusion. If uncertainty

arises, consult an official compendium, such as the Japanese Pharmacopoeia, Standard Methods of Analysis for Hygienic

Chemistry, Japanese Industrial Standards (JIS), which cover rules and regulations for the preparation of solutions.

(J.Ma)

1. Acetaminophen 2. Dihydrocodeine 3. Caffeine

Analytical Conditions

Column Shim-pack VP-ODS (1504.6 mm I.D.)

Mobile phase Top row A 9:1 (v/v) mixture of buffer solution and acetonitrile

Lower row

B

Make up to 10 % (v/v) acetonitrile with buffer solution.

The buffer solution is 20 mM sodium dihydrogen phosphate adjusted to pH 2.5 by the

addition of phosphoric acid.

Flowrate 1.0 mL/minute

Temperature 40 C

Detection 210 nm

Fig. 2 Effects of Mobile Phase Preparation Method


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Preparing the Mobile Phases SHIMADZU (Shimadzu Corporation).Pd

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