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4.
Operation steps
The following steps must be carried out under dark conditions
.
(1) Sample pretreatment
①Saponification : Take an appropriate amount of sample, equivalent to 1～5g of the original homogenate (containing 20～80ug of carotene ), the food sample depends on its carotene content, and the sampling amount of vegetable oil and high-fat samples should not exceed 10g
.
Place it in a 100 mL conical flask with stopper, add 30 mL of dealdehyde ethanol, then add 10 mL of potassium hydroxide solution (1+1), reflux for 30 minutes, and then quickly cool it with ice water
②Washing: Wash the saponified sample extract with water until it is neutral, filter it into a spherical bottle through a small funnel containing 10g of anhydrous sodium sulfate, and wash the separatory funnel and anhydrous sodium sulfate layer with a small amount of petroleum ether several times The pigment and washing liquid are incorporated into the spherical bottle
.
③Concentration and constant volume: The extract in the spherical bottle is distilled under reduced pressure on a rotary evaporator.
The temperature of the water bath is 60℃.
When it evaporates to about 1mL, remove the spherical bottle, dry it with nitrogen, and add 2.
00mL petroleum ether immediately.
Constant volume, ready for chromatography
.
(2) Paper chromatography
①Spotting: Make a baseline at the bottom of the 18cm×30cm filter paper 4cm away from the bottom edge.
Take four points A, B, C, and D on the baseline (see Figure 3-8), and draw 0.
100～0.
400mL concentrated solution in AB Quickly place samples between and CD
.
②Unfolding: After the spotted liquid on the paper evaporates naturally, the filter paper is rolled into a cylindrical shape and placed in a chromatography cylinder preliminarily saturated with petroleum ether for upward unfolding
.
③Elution: After the carotene is completely separated from other pigments, take out the filter paper, evaporate the petroleum ether naturally , cut off the carotene chromatography tape at the front of the developing agent , and immediately put it into a test tube with a stopper containing 5mL petroleum ether , Shake vigorously to completely dissolve the carotene in the solvent
.
Figure 3-8 Schematic diagram of paper chromatography spot
(3) Use a 1cm cuvette for measurement, adjust the zero point with petroleum ether , measure the absorbance at a wavelength of 450mm, and use its value to find the content of β-carotene from the standard curve for use in calculations
.
(4) Drawing the standard working curve.
Take the β-carotene standard use solution (concentration of 50ug/mL) 1.
5.
Result calculation
Where x——the content of carotene in the sample (calculated as β-carotene), mg/100g
m 1 ——The content of carotene found on the standard curve, ug
V 1 ——Spot volume, mL
V 2 ——Concentrated constant volume of sample extract, mL
m——sample mass, g
The calculation result retains three significant figures, and the absolute difference between the two independent measurement results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean
.
6.
Tips
(1) This method is simple and convenient, with clear color bands, and is suitable for the determination of carotene in plant foods and mixed foods containing plant foods
.
The detection limit of the method is 0.
(2) Anhydrous ethanol must not contain aldehydes.
The inspection method and de-aldehyde method are as follows:
① Inspection method: using silver mirror reaction method, that is, add concentrated ammonia water in 5% silver nitrate solution until the silver oxide precipitate is dissolved, add a few drops of 2.
5mol/L NaOH solution, if precipitation occurs, add concentrated ammonia water to dissolve it
.
Then add 2mL silver ammonia solution to the test tube, add a few drops of ethanol and shake well, add a little 2.
②Dealaldehyde method Take 2g of silver nitrate and dissolve in a small amount of water, take 4g of sodium hydroxide and dissolve in warm ethanol, pour the two into 1L ethanol, store in the dark for two days (shaking occasionally to promote the reaction), filter, and pour the filtrate Distill in a distillation flask, discard the 50mL that was first steamed
.
When ethanol contains more aldehydes, the amount of silver nitrate can be increased appropriately
(3) Normally β-carotene standards cannot be completely dissolved in organic solvents.
If necessary, the standards should be saponified first, then extracted with organic solvents, washed with distilled water to neutrality, concentrated to a constant volume, and then calibrated
.
Since carotene is easy to decompose, the standards used must be calibrated before each use, and the samples should be measured simultaneously with the standards
(4) extract was concentrated to dryness must prevent, or avoid carotene oxidation due to high temperature, ultraviolet radiation and other damage in the air
.
Operations such as constant volume, spotting, and cutting sample points after chromatography must also be quick
.
Related Links: Determination of Carotene in Food (1)
