LU503193B1 - Method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography - Google Patents
Method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography Download PDFInfo
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- LU503193B1 LU503193B1 LU503193A LU503193A LU503193B1 LU 503193 B1 LU503193 B1 LU 503193B1 LU 503193 A LU503193 A LU 503193A LU 503193 A LU503193 A LU 503193A LU 503193 B1 LU503193 B1 LU 503193B1
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- monosaccharides
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- cell wall
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8836—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving saccharides
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Abstract
The invention discloses a method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography, which comprises the following steps: firstly, extracting polysaccharides in apple pulp cell wall and hydrolysing with acid, then carrying out enzymolysis, monosaccharides derivatization, and finally exposing samples in a sample bottle to the high performance liquid chromatography for detection. The monosaccharides obtained by the method of the invention can be stored in a short time before derivatization, which is more practical compared with the time requirement of gas chromatography and relatively more accurate in determination. The optimization method in the invention can be used for detecting monosaccharides in apple pulp cell wall. The apple sample in the detection method of the invention only has a hydrolysis process, and monosaccharides derivatives in the sample after hydrolysis are more stable and more accurate than those in gas chromatography; what’s more, when the HPLC method is used for monosaccharides composition analysis, it has the advantages of high separation speed, high resolution, good reproducibility and the like; and by adopting the detection method of the invention, monosaccharides in apple pulp cell walls can be directly analysed by using the HPLC method combined with a universal detector.
Description
1 LU503193
METHOD FOR DETECTING MONOSACCHARIDES IN APPLE PULP
CELL WALL BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
The invention belongs to a method for detecting monosaccharides in apple pulp cell wall, and particularly relates to a method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography.
In the prior art, the determination methods of monosaccharides composition of polysaccharides mainly include thin layer chromatography, gas chromatography, high performance capillary electrophoresis and high performance liquid chromatography (HPLC). Thin-layer chromatography has the characteristics of convenient operation, simple equipment and no need for derivatization, but its sensitivity is low and its separation effect is poor, so it is only suitable for the qualitative analysis of simple polysaccharides. Gas chromatography has high separation efficiency, rapid determination and high sensitivity, but saccharides and polyols have low volatility, thermal stability and high boiling point, which need derivatization before they can be analysed by gas chromatography. However, the reaction operation of monosaccharides derivatives by gas chromatography is very complex, which is easy to produce isomers and easily interfered by sample impurities, leading to large errors. Amperometric detection by high performance anion exchange chromatography is an ideal method for technical analysis of saccharides. It takes advantage of the electrochemical activity of saccharides, and monosaccharides can be ionized in strong alkali solution. Therefore, strong alkali solution can be used as mobile phase for anion exchange to achieve separation effect. However, the general-purpose differential refractive index detector has low sensitivity and high detection limit, while the evaporation light scattering detector, another general-purpose detector, is expensive and has complicated detection steps.
Monosaccharides molecules themselves lack chromophoric groups, so they can't directly use ultraviolet or fluorescence detectors, which limits the analysis and detection of monosaccharides to some extent. Some studies have derived monosaccharides after
2 LU503193 hydrolysis of polysaccharides with luminescent reagents, and then separated and detected them by HPLC [13-17]. The principle is that 1-phenyl-3-menthy-5-pyrazolone (PMP) can be quantitatively condensed with monosaccharides under alkaline conditions to form
PMP-monosaccharides derivatives, which are stable and have strong light absorption at wavelength of 250nm [18-20] so that ultraviolet detector can be applied to the analysis of monosaccharides. Among them, pre-column high performance liquid chromatography (HPLC) has been widely used because of its fast, accurate and simple method, and has been applied to the analysis of monosaccharides composition of various polysaccharides, but the HPLC of conventional acid hydrolysis pre-column derivatization method in the prior art cannot be applied to apple pulp. Based on the above defects in the prior art, it is urgent to design a method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography.
In order to overcome the defects in the prior art, the invention provides a method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography.
The invention is realized by the following technical scheme: the invention discloses a method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography, which specifically comprises the following steps: (1) firstly, extracting the cell wall polysaccharides of apple pulp;
Acid hydrolysis: weighing 10mg of apple pulp polysaccharides AIR samples each, three repetitions in one group, three groups in total, putting them into a glass tube, adding 3ml of 2mol/l trifluoroacetic acid, adding nitrogen, sealing them, shaking them in a constant temperature water bath shaker at 70 ~ 90°C for 65 ~ 75 hours, then taking them out and adjusting the PH value; (2) Then moving on to enzymolysis:
Adding 25ul VLO reverse transcriptase into the solution, heating the solution to 45 ~ 55°C, shaking it in a shaker for 20 ~ 26h, taking it out and boiling it for 12 ~ 20 minutes after shaking; (3) monosaccharides derivatization:
3 LU503193
Taking 100ul of the liquid obtained in step (2), adding 100ul of sodium hydroxide into the liquid, then adding 200ul 0.6mol/L PMP, mixing evenly, performing water bath the mixed liquid at 65°C ~ 75°C for 90 minutes ~ 110 minutes, taking it out and keeping it away from light for 8 minutes ~ 12 minutes, then adding 100ul of hydrochloric acid and mixing evenly; Adding chloroform for extraction for 2-3 times, taking supernatant, passing through a membrane, and putting into a sample bottle; (4) exposing the sample in the sample bottle to high performance liquid chromatography for detection:
The detection conditions of HPLC are as follows: mobile phase phosphate buffer with pH 6.85 is applied, gradient elution with acetonitrile is carried out, and column temperature of HPLC is set at 30°C ~ 40°C.
Preferably, when PH is adjusted, the PH value of the solution is adjusted to 5.0 with 1mol/L NaOH solution and 0.1mol/L NaOH solution respectively. In step (1), it is shaken at 80°C for 72 hours in a constant temperature water bath shaker.
Preferably, in step (2), the solution is heated to 50°C and shaken in a shaker for 24 hours. After shaking, it is taken out and boiled for 15 minutes.
Preferably, in step (3), the mixed solution is put in a water bath at 70°C for 100 minutes, and is taken out and kept away from light for 10 minutes and chloroform is added for extraction for three times.
Preferably, in step (4), the column temperature of the high performance liquid chromatograph is set at 35°C.
The invention has the beneficial effects that the invention provides a method for detecting monosaccharides in apple pulp cell walls by high performance liquid chromatography, which is intended for measuring monosaccharides such as rhamnose, glucose, arabinose, xylose, fucose, mannose and galactose in pectin by more and more common pre-column derivatization methods at present, but common acid hydrolysis methods such as trifluoroacetic acid and sulfuric acid in the prior art can not hydrolyse pectin polysaccharides in apple pulp into monosaccharides, and the optimized method of the invention can be used to obtain monosaccharides hydrolysed by apple pulp polysaccharides. The enzymolysis step in the invention is the feature and key step of the
4 LU503193 method, which 1s a key technical point distinguishing from the existing technical scheme in the prior art. The monosaccharides obtained by the method can be stored in a short time before derivatization, which is more practical compared with the time requirement of gas chromatography and relatively more accurate in determination. The optimization method in the invention can be used for detecting monosaccharides in apple pulp cell wall. The apple sample in the detection method of the invention only has a hydrolysis process, and monosaccharides derivatives in the sample after hydrolysis are more stable and more accurate than those in gas chromatography; secondly, when the HPLC method is used for monosaccharides composition analysis, it has the advantages of high separation speed, high resolution, good reproducibility and the like; and by adopting the detection method of the invention, monosaccharides in apple pulp cell walls can be directly analysed by using the HPLC method combined with a universal detector.
Fig. 1 to Fig. 5 are chromatograms for detecting monosaccharides in different apple pulp cell walls by using the high performance liquid chromatography of the present invention.
The present invention will be described in detail with reference to specific embodiments.
The invention discloses a method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography, which comprises the following steps: (1) Firstly, extracting the cell wall polysaccharides of apple pulp: acid hydrolysis: weighing 10mg of apple pulp polysaccharides AIR samples each, three repetitions in one group, three groups in total, putting them into a glass tube, adding 3ml of 2 mol/l trifluoroacetic acid, adding nitrogen, sealing them, shaking them in a constant temperature water bath shaker at 80°C for 72hours, then taking them out and adjusting the PH value as 5.0 with with 1mol/L NaOH solution and 0.1mol/L NaOH solution respectively;(2)Then moving on to enzymolysis: adding 25ul VL9 reverse transcriptase into the solution, heating the solution to 50°C, shaking it in a shaker for 24h, taking it out and boiling it for 15 minutes after shaking; (3)monosaccharides derivatization: taking 100ul of the liquid obtained in step (2), adding 100ul of sodium hydroxide into the liquid, then adding 200ul 0.6mol/L of PMP, mixing evenly, performing water bath the mixed liquid at 70°C for 100 minutes, taking it out and keeping it away from light for 10 5 minutes, then adding 100ul of hydrochloric acid and mixing evenly; Adding chloroform for extraction 3 times, taking supernatant, passing through a membrane, and putting into a sample bottle; (4) Exposing the sample in the sample bottle to high performance liquid chromatography for detection:
The detection conditions of HPLC are as follows: mobile phase phosphate buffer with pH 6.85 is applied, gradient elution with acetonitrile is carried out, and column temperature of HPLC is set at 35°C.
Finally, it should be noted that the above contents are only used to illustrate the technical scheme of the present invention, but not to limit the protection scope of the present invention. Simple modifications or equivalent substitutions made by ordinary technicians in the field do not depart from the essence and scope of the technical scheme of the present invention.
Claims (8)
1. A method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography, comprising; (1) extracting the cell wall polysaccharides of apple pulp; acid hydrolysis: weighing 10mg of apple pulp polysaccharides AIR samples each, three repetitions in one group, three groups in total, putting into a glass tube, adding 3ml of 2 mol/l trifluoroacetic acid, adding nitrogen, sealing, shaking in a constant temperature water bath shaker at 70 ~ 90°C for 65 ~ 75 hours, then taking out and adjusting the pH value; (2) enzymolysis: adding 25ul VLO reverse transcriptase into the solution, heating the solution to 45 ~ 55°C, shaking in a shaker for 20 ~ 26h, taking out and boiling for 12 ~ 20 minutes after shaking; (3) monosaccharides derivatization: taking 100ul of the liquid obtained in step (2), adding 100ul of sodium hydroxide into the liquid, then adding 200ul 0.6mol/L PMP, mixing evenly, performing water bath the mixed liquid at 65°C ~ 75°C for 90 minutes ~ 110 minutes, taking out and keeping away from light for 8 minutes ~ 12 minutes, then adding 100ul of hydrochloric acid and mixing evenly; adding chloroform for extraction for 2-3 times, taking supernatant, passing through a membrane, and putting into a sample bottle; (4) putting the sample in the sample bottle to high performance liquid chromatography for detection: the detection conditions of HPLC are as follows: mobile phase phosphate buffer with pH 6.85 is applied, gradient elution with acetonitrile is carried out, and column temperature of HPLC is set at 30 ~ 40°C.
2. The method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography according to claim 1, wherein that in step (1), when PH is adjusted, the PH value of the solution is adjusted to 5.0 with 1mol/L NaOH solution and 0.1mol/L NaOH solution respectively.
7 LU503193
3. The method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography according to claim 1 or 2, wherein in step (1), it is shaken at 80°C for 72 hours in a constant temperature water bath shaker.
4. The method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography according to claim 1 or 2, wherein in step (2), the solution is heated to 50°C and shaken in a shaker for 24 hours.
5. The method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography according to claim 1 or 2, wherein in step (2), after shaking, it is taken out and boiled for 15 minutes.
6. The method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography according to claim 1 or 2, wherein in step (3), the mixed solution is put in a water bath at 70°C for 100 minutes, then is taken out and kept away from light for 10 minutes.
7. The method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography according to claim 1 or 2, wherein in step (3), chloroform is added for extraction for three times.
8. The method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography according to claim 1 or 2, wherein in step (4), the column temperature of the high performance liquid chromatography is set at 35°C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU503193A LU503193B1 (en) | 2022-12-15 | 2022-12-15 | Method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU503193A LU503193B1 (en) | 2022-12-15 | 2022-12-15 | Method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography |
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| LU503193B1 true LU503193B1 (en) | 2023-06-15 |
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| Application Number | Title | Priority Date | Filing Date |
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| LU503193A LU503193B1 (en) | 2022-12-15 | 2022-12-15 | Method for detecting monosaccharides in apple pulp cell wall by high performance liquid chromatography |
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- 2022-12-15 LU LU503193A patent/LU503193B1/en active IP Right Grant
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