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[Readers Insight] When to Use Acetone as a Mobile Phase?
This article is written by Welch's contract writer Chromatography Mound. The content of the article presents a point of view from the author solely.
Table of contents
Introduction
Acetone is a common solvent in analytical testing, recognized for its exceptional dissolving power. It effectively dissolves various organic substances, fats, rubbers, and modified celluloses. Furthermore, acetone acts as a versatile "multitasker" for polarity adjustment, as it is miscible with solvents of varying polarities, including water, methanol, ethanol, ethyl ether, dichloromethane, chloroform, pyridine, and n-hexane.
While it is a staple in sample pretreatment, acetone is rarely utilized during the preparation of mobile phases. In this article, we will explain the reasons for the limited use of acetone, and when it should be used.
Practical Applications of Acetone in Mobile Phases
Acetone is specified in certain regulatory methods. For example, in the Chinese national standard GB/T 19681-2005 (Method for the determination of Sudan dyes in foods -- High performance liquid chromatography), acetone is utilized in the mobile phase. In the later GB/T 29663-2013 (Determination of Sudan I, II, III, IV in cosmetics -- High performance liquid chromatography), acetone is also adopted as the organic phase in the mobile phase.
The reason for using acetone in these standards will be explained in the final chapter.
7.2.1 Instrumental conditions:
- Column: Zorbax SB-C18 3.5 µm, 4.6 mm × 150 mm (or equivalent)
- Mobile Phase:
Phase A: 0.1% formic acid in water / acetonitrile (85:15)
Phase B: 0.1% formic acid in acetonitrile / acetone (80:20) - Gradient program:
Flow rate: 1 mL/min
Column temperature: 30 °C
Wavelength: Sudan I: 478 nm; Sudan II, III, IV: 520 nm; switch after Sudan I exhibits peak.
Injection volume: 10 µL
Gradient profile (omitted)
Instrumental conditions in GB/T 19681-2005
Why Is Acetone Rarely Used?
The answer is simple. The primary reason acetone is rarely used as the organic phase in reversed-phase chromatography is that methanol and acetonitrile satisfy the vast majority of elution and separation requirements.
The essence of elution involves modifying the polarity of the mobile phase by adjusting the ratios of its components. When methanol or acetonitrile is mixed with water in any proportion, the resulting polarity range is sufficient to cover the separation needs of most compounds, rendering the use of acetone unnecessary in most cases.
When Do We Use Acetone?
When using acetone as a mobile phase, the following conditions should be met:
1. The proportion should not be too high
Acetone has relatively low polarity and stronger elution power than acetonitrile. When using acetone as the organic phase, the proportion of acetone should not be too high.
If the analyte is highly polar or moderately polar, acetone is often not suitable as the organic phase because the compounds may elute from the column too quickly to achieve good separation.
2. The detection wavelength should be higher than 330 nm
The UV cutoff wavelength of acetone is approximately 330 nm. When the detection wavelength is lower than this value, strong background absorption may occur, leading to:
- elevated baseline
- increased noise
- masking of the target peak
3. Pay attention to the effects on the column and instrument
High proportions of acetone, or long-term use of acetone, may:
- damage chemical bonds in the stationary phase;
- cause loss of the stationary phase;
- shorten column lifetime;
- cause swelling or damage to polymer materials in pump seals, proportioning valves, and similar components.
Therefore, when acetone is used as a mobile phase, not only should its proportion be controlled, but the column and system should also be flushed with methanol after use to remove residues.
Example: Use of Acetone in Sudan Dye Analysis
Now we explain why acetone is used in the analysis of Sudan dyes. Sudan I, II, III, and IV are compounds of extremely low polarity that may precipitate if the water proportion in the mobile phase is too high. Additionally, they exhibit extremely strong retention on conventional C18 columns, making them difficult to elute without a low-polarity organic phase.
In these specific cases, even high proportions of methanol or acetonitrile may fail to provide a sufficient polarity gradient to achieve effective separation. This often results in situations where a mixed standard displays a clear chromatogram, yet actual samples fail to show adequate resolution between target analytes and impurities.
Furthermore, the maximum absorption wavelength of Sudan dyes is above 450 nm, which satisfies the requirement that the detection wavelength remains above the UV cutoff of acetone.
| Compound | CAS No. | Chemical Name | Formula | Rel. Molecular Mass | Structure |
|---|---|---|---|---|---|
| Sudan I | 842-07-9 | 1-(Phenyldiazenyl) naphthalen-2-ol | C16H12ON2 | 248.28 |  |
| Sudan II | 3118-97-6 | 1-(2,4-Dimethylphenylazo)-2-naphthol | C18H16ON2 | 276.34 |  |
| Sudan III | 85-86-9 | 1-{[4-(Phenyldiazenyl)phenyl] diazenyl}naphthalen-2-ol | C22H16ON2 | 352.4 |  |
| Sudan IV | 85-83-6 | 1-[{2-Methyl-4-[(2-methylphenyl) diazenyl]phenyl}diazenyl] naphthalen-2-ol | C24H20ON2 | 380.45 |  |
Conclusion
In summary, the utilization of acetone as an organic component in HPLC mobile phases is characterized by stringent operational requirements and is often unnecessary for routine analytical procedures. Its application remains specialized, primarily restricted to specific cases such as the determination of Sudan dyes, where its unique chemical properties are essential for achieving effective separation and elution.