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【 Application 】 Using SFC to separate the main extract of saffron
Date: 2025-09-11Read: 2
Büchi Labortechnik AG
Using SFC to separate the main extract of saffron
SFC application
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Introduction
Saffron is an expensive spice. Although the flowers of saffron plants have a delicate purple hue, their filamentous red stigma is highly valuable as it can be used as a spice and natural dye. In autumn, the stigma is hand picked and separated to produce red spices. Producing one pound (0.45 kilograms) of saffron requires 70000 flowers.
Saffron, its glycoside derivatives, and crocetin are compounds that contribute to the color and taste of saffron. Safranal， A monoterpenoid aldehyde also exists. In addition, there are some compounds in saffron that have pharmacological activity, such as crocin derivatives and flavonoids. For example, saffron carotenoids have been proposed as alternative anti-tumor drugs and may be used alone or in combination with other compounds to treat certain cancers in the future.
Therefore, there are many publications dealing with the extraction and analysis of saffron literature. Usually, reverse phase liquid chromatography (RP-LC) is used for separation. Typical solvents are water/acetonitrile or water/methanol mixtures. As usual, the stationary phase is C18. The initial separation condition is high water content, and the gradient method is used to significantly increase the organic solvent content over time to elute non-polar compounds. Formic acid is often added to the mobile phase.
Supercritical fluid chromatography (SFC) is a technique that utilizes supercritical carbon dioxide (CO)2）As a chromatographic method for mobile phase. Supercritical carbon dioxide hasHigher diffusion coefficient and lower viscosityIt is an excellent solvent for separating and analyzing compounds. Compared to other types of chromatography, SFC provides several advantages, includingFaster analysis time, lower solvent usage, and different selectivityCompared to RP-LC, SFC is an orthogonal technique.
This application manual introduces a method for separating the main components of saffron thread using SFC and mass spectrometry. The ionization of analytes is performed by electron spray ionization (ESI). Due to the use of CO in the mobile phase2 Compared to methanol, it has slight acidity and therefore does not require the use of additives.
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Equipment
Sepiatec SFC Instrument
Advion CMS detector
Nucleodur NH2 5 μm 250 x 4 mm
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Reagents and materials
Carbon dioxide (99.9%)
Methanol (≥ 99%)
Saffron thread
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Experimental Procedure
Sample preparation:
1000mg saffron was extracted 5 times with 10mL of hot methanol. The final solution is filtered for the separation of natural compounds.
Experimental conditions:
Mobile phase |
A=Carbon dioxide; B=Methanol |
Mobile phase condition |
0-1 minute: 14% B |
1-18 minutes |
14 - 18% B |
18-40 minutes |
18 - 50% B |
40-44 minutes |
50% B |
detection |
UV 440 nm MS ESI （+/-） |
Under the conditions of 86/14% supercritical carbon dioxide and methanol, a flow rate of 7 mL/min was used to treat Nucleodur NH2Balance at 5 μ m and 250 × 4 mm for 5 minutes. Use an automatic sampler to inject (V=100 uL) and start running (running time=44 min). Set the back pressure regulator to 150 bar and heat the column box to 40 ° C.
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Experimental results and discussion
▲ Figure 1. UV chromatogram of saffron extract after purification (UV wavelength 440 nm)
The main components of saffron were extracted using methanol. Due to methanol being a polar solvent, it can dissolve some polar compounds.Figure 1The UV chromatogram at 440 nm. In the first 18 minutes, due to the mobile phase (86-82% CO)2）It has non-polar characteristics and no UV active compound elution. When the polarity of the mobile phase increases through a gradient, several polar compounds are eluted. Due to UV detection not allowing identification of components, SFC coupled with MS can be continuously identified.Figure 2For UV and mass spectrometry images. MS signals are based on different qualities: (b) mass 999-999.5 (ESI+); (c) mass 836.9 – 837.4 (ESI+); (d) mass 674.8 – 675.3 (ESI+); (e) mass 975.5 – 976 (ESI-); (f) mass 813.4 – 813.9 (ESI-); (g) mass 651.4 – 651.9 (ESI-) and (h) mass 341.2 – 341.7 (ESI-).
▲ 图2. (a) UV- and (b – h) MS-Chromatograms of the purification of saffron extract; (b) mass 999 – 999.5 (ESI+); (c) mass 836.9 – 837.4 (ESI+); (d) mass 674.8 – 675.3 (ESI+); (e) mass 975.5 – 976 (ESI-); (f) mass 813.4 – 813.9 (ESI-); (g) mass 651.4 – 651.9 (ESI-) and (h) mass 341.2 – 341.7 (ESI-)
Table 1forFigure 2The main compounds identified by mass spectrometry. ESI ionization of molecules is used in mass spectrometry analysis, which is a mild ionization method at atmospheric pressure. Ionization can be carried out under positive voltage (ESI+) or negative voltage (ESI -). In the positive ion mode, sodium adducts ([M+Na]+) or proton adducts ([M+H]+) are usually formed. In negative ion mode, ([M-H] -) ions are typically formed due to the loss of a proton. According to the different samples and their properties, various charged products can also be formed.
Saffron combines with several sugars (glucose, gentian disaccharides, triglycerides, and neoglycosides) to form crocetin. The strong increase in the polarity of the covalent bond between crocetin and sugar molecules makes crocetin hydrophilic, which is why the main component identified later in the AN eluent. fromTable 1It can be seen that there is a binding of crocetin with gentian and glucose in saffron silk thread.
The compound ketoxin disulfide ester is composed of ketoxin and two gentian sugar molecules, which are constructed from two D-glucose molecules. InFigure 2 (b)The compound was identified as a positive ion with a mass of 999-999.5. The adduct consists of sodium (mass 23 g/mol) and sample molecules (mass 976.4 g/mol). The corresponding spectrum is as followsFigure 3 (a)As shown. Sweet potato glycosides can also be detected under negative ionization voltage（Figure 2 (e)Quality 975.5-976), corresponding mass spectra as shown inFigure 3 (b)As shown.
In ESI+mode（Figure 2 (c)And ESI mode（Figure 2 (f)）The biobased glycosyl esters of saffron root are ([M+Na]+mass 836.9~837.4) and ([M - h] - mass 813.4~813.9), respectively. The corresponding mass spectrum is shown asFigure 3 (c) and (d)As shown. Compound crocetin gentian bioglycosyl ester is composed of crocetin, gentian sugar, and D-glucose molecules.
In ESI+mode（Figure 2 (d)And ESI mode（Figure 2 (g)）Below, the gentiobiosyl esters of crocin are ([M+Na]+mass 674.8-675.3) and ([M-H] - mass 651.4-651.9), respectively. The corresponding mass spectrum is as followsFigure 3 (e) and (f)As shown. Crocetin methyl ester can only be recognized in ESI mode（Figure 2 (h)Quality 341.2-341.7 andFigure 3 (g)）.
In the ESI process, the sample molecules are also fragmented, which is why in Figure 2, some saffron is assigned to multiple masses. For example, guava dithiocarbamate can ionize into adducts ([M+Na]+) or ([M-H] -), but can also be further broken. During the negative ion process, the removal of glucose groups results in fragments with a quality similar to that of crocin root biogenic glycosides and crocin root biogenic esters.
▲ Table 1. Structure and molecular weight of the main components identified by mass spectrometry in saffron silk thread (Figure 2).
Compound 1 underwent mass directed fractionation in both ESI+and ESI - modes, while compound 2 underwent mass directed fractionation only in ESI+mode. Compound 4 can only be collected in ESI mode. However, the extraction mass spectrum of compound No. 3 became complex due to the presence of fragments with similar structures to compounds 1 and 2, which interfered with the mass directed fractionation process. To address this issue, an additional time window was adopted.
▲Figure 3. Mass spectra of crocin disaccharide esters (a) ESI+and (b) ESI -, crocin root biogenic glycosyl esters (c) ESI+and (d) ESI -, crocin root biogenic esters (a) ESI+and (b) ESI - and (g) crocin methyl ester ESI -
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Experimental Conclusion
When analyzing natural products, unknown products often dissolve during the extraction process. To identify these unknown components, mass spectrometry analysis is required. This study used methanol to extract the main components of saffron thread and analyzed them using SFC-UV/MS. The main components were continuously identified using mass spectrometry.