《ISO-5510-1984.pdf》由会员分享，可在线阅读，更多相关《ISO-5510-1984.pdf（7页珍藏版）》请在三一文库上搜索。
1、International Standard INTERNATlONAL ORGANIZATION FOR STANDARDIZATION*MEWlYLYHAPOHAR OPTAHH3AUMfl no CTAHAPTM3Al 8 g of sodium hydroxide; 16 ml of hydrochloric acid (ezo = I,19 g/ml); 0,l ml of octanoic (caprylic) acid; 20 ml of thiodiglycol; 3 ml of a 30 % ( V/ V) aqueous solution of polyoxyethylen
2、e dodecyl ether with approximately 23 molecules of oxy- ethylene.2) Dilute to 1 000 ml with water. 2) A suitable commercially available product is BRIJ 35. This information is given for the convenience of the user of this international Standard and does not constitute an endorsement of this product
3、by ISO. 1 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/27/2007 09:21:27 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 5510-1964 1E) 5.6 Sodium citrate,
4、 buffer solution, pH 5,28. Successively dissolve in water 24,5 g of citric acid monohydrate; 14 g of sodium hydroxide; 6,8 ml of hydrochloric acid (0 = I,19 g/ml); 0,l ml of octanoic (caprylic) acid; 3 ml of a 30 % (V/v) aqueous solution of polyethoxyl dodecyl ether with approximately 23 molecules o
5、f oxy- ethy1ene.l Dilute to 1 000 ml with water. Adjust the pH to 5,28 f 0,02 using hydrochloric acid (ezO = I,19 g/ml) or 50 % (m/m) sodium hydroxide solution. 5.7 Ninhydrin reagent. 5.7.1 Sodium propionate, buffer solution, pH 5,5 + 0,l. Dissolve 168 g of sodium hydroxide in about 400 ml of water.
6、 Cool, then add while shaking, 498 ml of propionic acid. Dilute to 1 000 ml with water. 5.7.2 Preparation of reagent. Prepare the ninhydrin reagent in an atmosphere of nitrogen and in the dark. Into a 2 I flask, place 1 litre of peroxide-free ethylene glycol monomethyl ether, 1 litre of the sodium p
7、ro- pionate buffer solution (5.7.1) and 40 g of ninhydrin. Shake un- til completely dissolved, then add I,33 g of tin(ll) chloride dihydrate (SnC12.2H20). Shake until completely dissolved. (See the note.) This reagent is stable for 1 month if kept at 4 C under light pressure in an atmosphere of nitr
8、ogen and in the dark. NOTE - If precipitation of SnCl2 occurs, replace the tinIll) chloride with 7,5 ml of 150 g/l titanium (ill) chloride solution or by 1.5 g of hydrindantin per litre of reagent. 5.8 Lysine, standard solution, corresponding to 1 mmol of lysine base per litre. Dissolve 182,5 mg of
9、lysine monohydrochloride in 100 ml of 0.1 mol/l hydrochloric acid. Take exactly 10 ml of this solution and dilute it to 100 ml with sodium citrate buffer solution of pH 2,2 (5.5). 1 ml of this solution contains 1 pmol of lysine base. 6 Apparatus Usual laboratory equipment, and in particular: 6.1 Gri
10、nder, having the following characteristics: a) constructed of a material which does not absorb moisture; b) easy to clean and having minimum dead space; c) permitting rapid and uniform grinding, without causing undue heating and preventing, as much as possible, contact with the outside air; d) capab
11、le of being regulated to give particles of the sizes specified in 7.1. 6.2 Flasks, of capacities 250 ml and 1 000 ml, flat- bottomed, short-necked with ground conical joints and reflux condensers to fit the flasks. 6.3 Crucible, with a sintered glass plate of grade P 16 (pore size index 10 to 16 pm)
12、. 6.4 Oil-bath, capable of being maintained at a temperature which will ensure refluxing (120 to 130 “C). 6.5 Rotary evaporator. 6.6 Apparatus for automatic analysis of amino acids, or if this is not available, manual chromatographic system comprising : a) a chromatography column, of length about 25
13、0 mm and of internal diameter 6 mm, thermostatically maintained at 55 OC by means of a water jacket and circulation bath, and filled up to 200 mm with an 8 % cross-linked strongly acidic, cation exchange resin with sulfonic functional groups, of particle size 13 f 2 VrnY b) small piston pump, produc
14、ing a flow of 50 ml/h; c) fraction collector; d) boiling water-bath; e) spectrometer, set at 570 nm, with cells of thickness 10 mm. 6.7 Graduated pipettes, of capacities 1; 5; and 10 ml. 6.8 Volumetric flasks, of capacities 10; 20; and 100 ml. 6.9 Analytical balance. 7 Sampling See IS0 6497. 1) A su
15、itable commercially available product is BRIJ 35. This information is given for the convenience of the user of this International Standard and does not constitute an endorsement of this product by ISO. 2) A suitable commercially available product is Aminex A5. This information is given for the conve
16、nience of the user of this International Standard and does not constitute an endorsement of this product by ISO. 2 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/27/2007 09:21:27 MDTNo
17、reproduction or networking permitted without license from IHS -,-,- IS0 5510-1984 (E) 8 Procedure of the lysine standard solution (5.811 or the amount specified in the manufacturer s instructions. 8.1 Preparation of the test sample Grind 5 to 10 g of the laboratory sample to obtain particles which w
18、ill pass completely through a sieve of aperture size 315 urn. NOTE - The aperture size specified is smaller than that recommended in IS0 6498 in order to ensure maximum contact with the DNFB. 8.2 Test portions Weigh, to the nearest 1 mg, and place respectively in the two flasks (6.2), two test porti
19、ons each of which corresponds to approximately 100 mg of crude protein. (See IS0 5983 for the determination of the protein content.) 8.3 Total lysine 8.3.1 Hydrolysis with hydrochloric acid Into the 1 000 ml flask (see 8.21, place 500 ml of the 6 mol/l hydrochloric acid (5.4). Fit a reflux condenser
20、 and place the flask in the oil-bath (6.4), previously heated to 120 to 130 Y. After boiling gently for 24 h, cool the flask and filter the con- tents through the crucible (6.3). Evaporate the filtrate at a tem- perature not exceeding 40 OC, using the rotary evaporator (6.5). Add water to the residu
21、e thus obtained and evaporate. Repeat this operation until most of the hydrochloric acid has been removed; in general, four rinses with 30 ml of water are sufficient. Dissolve the residue in sodium citrate buffer solution of pH 2,2 (5.5) and transfer quantitatively to a 100 ml one-mark volumetric fl
22、ask (6.8). Make up to the mark with the sodium citrate buffer solution of pH 2,2 (5.5). Filter through a folded filter paper. 8.3.2 Final preparation of the column and adsorption of the hydrolysate Connect the pump of the apparatus (6.6) to a reservoir contain- ing sodium citrate buffer solution of
23、pH 5,28 (5.6); then adjust it to obtain a flow of 50 ml/h. Connect the pump to the resin column, previously heated to 55 OC, and allow the buffer solu- tion (5.6) to pass through the column for 20 min in order to establish equilibrium. Disconnect the pump. Remove most of the liquid above the resin,
24、taking care that the surface of the resin does not become dry. By means of a pipette (6.71, take 0,5 ml of hydrolysate (or 1 ml if using the manual system), place it on the column, then allow it to pass through the resin with the aid of a slight nitrogen pressure. Rinse the walls of the column with
25、0,5 ml of sodium citrate buffer solution of pH 5,28 (5.6) and pass through the resin. Fill the column to the top with sodium citrate buffer solu- tion (5.6) and connect it to the pump. 8.3.3 Determination 8.4 Non-available lysine 8.3.3.1 Automatic system 8.4.1 Dinitrophenylation reaction Set the ana
26、lyser in operation. Calibrate the apparatus, pro- ceeding as described in 8.3.2 using 0,25 umol of lysine IO,25 ml 8.3.3.2 Manual system 8.3.3.2.1 Localization of lysine The fraction of eluate containing the lysine shall be checked by using a lysine so!ution of known concentration, for example the 1
27、 umol/ml solution (5.8). For this purpose, discard the first 25 to 30 ml of eluate and then collect fractions of 1 ml in test- tubes until the 50th fraction is obtained, reckoned from the beginning of the elution. Add to each fraction, between the 30th and the 50th fractions, 1 ml of the ninhydrin r
28、eagent (5.7). Mix, transfer to the boiling water-bath and leave for 15 min. Cool. Dilute by adding 10 ml of sodium citrate buffer solution of pH 5,28 (5.6). Mix and measure the absorbance at 570 nm, by means of the spectrometer, using sodium citrate buffer solu- tion of pH 5,28 (5.6) as the referenc
29、e liquid. NOTE - For information only, under the conditions described, lysine generally elutes between the 39th and 45th fractions. 8.3.3.2.2 Determination If the lysine is eluted between the 39th and 45th fractions discard the first 38 ml of eluate, collect and combine the frac- tions (Nos 39 to 45
30、) corresponding to the lysine peak and evaporate using the rotary evaporator (6.5). NOTE - After recovering the fractions containing the lysine, allow about 200 ml of buffer solution to pass through the column in order to remove any undesirable components which may remain. Dissolve the residue in 5
31、ml of the sodium citrate buffer solu- tion of pH 5,28 (5.6) and add 5 ml of the ninhydrin reagent (5.7). Mix, transfer to the boiling water-bath and leave for 15 min. Cool. Dilute the mixture with the sodium citrate buffer solution of pH 5,28 (5.6) so that the lysine concentration of the test soluti
32、on is approximately 0,02 umol/ml. (Let VI be the volume of test solution thus obtained.) Measure the absorbance at 570 nm by means of the spec- trometer, using as the reference liquid a mixture of one part by volume sodium citrate buffer solution (5.6) and one.part by volume ninhydrin reagent (5.71,
33、 which has been placed for 15 min in the boiling water-bath and diluted to the volume V, after cooling. +r: -813.3.2.3 Calibration of the spectrometer i- Take exactly 5 ml of the standard lysine solution (5.8). Add 5 ml of the ninhydrin reagent (5.7). Mix, transfer to the boiling water-bath and leav
34、e for 15 min. Cool and dilute to 100 ml with sodium citrate buffer solution of pH 5,28 (5.6). Measure the absorbance, by means of the spectrometer, under the same conditions as in 8.3.3.2.2. By means of a pipette (6.71, transfer 8 ml of the sodium bicar- bonate solution (5.2) to the 250 ml flask (se
35、e 8.2). Leave for 3 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/27/2007 09:21:27 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 5510-1994 (E) about 10
36、min, stirring from time to time. Then add the DNFB solution (5.3). stopper the flasks, agitate, and ensuring that there are no particles adhering to the walls of the flask, leave the mixture overnight at ambient temperature and in darkness. 8.4.2 Purification Evaporate to dryness at a temperature be
37、low 40 OC, using the rotary evaporator (6.5). Add 75 ml of the diethyl ether (5.1) to the flask, stir and allow to separate. Pour off most of the, diethyl ether, taking care to avoid entraining solid particles with it. Repeat these operations twice more, using each time 50 ml of the diethyl ether. E
38、vaporate the last traces of diethyl ether by heating on the rotary evaporator. -. 8.4.3 Hydrolysis with hydrochloric acid Transfer to the flask 150 ml of the hydrochloric acid (5.4) and proceed as described in 8.3.1 but transfer quantitatively the residue, dissolved in sodium citrate buffer solution
39、 of pH 2,2 (5.5) to a 20 ml volumetric flask (6.8) (or 10 ml if a manual system is being used). 8.4.4 Deposition of the hydrolysate and determination V, is the volume, in millilitres, of hydrolysate transferred to the column (generally, Vc = 0,5 ml). 9.1.1.2 Determination using a manual system The t
40、otal lysine, w, expressed as a percentage by mass, is equal to v, x 0,073 A, x- ml x vo A0 where A0 is the absorbance of the standard lysine solution deter- mined in 8.3.3.2.3; A, is the absorbance determined in 8.3.3.2.2; V. is the volume, in millilitres, of hydrolysate transferred to the column (g
41、enerally, V. = 1 ml); VI is the volume, in millilitres, of the test solution; ml is the mass, in grams, of the test portion placed in the first flask. Proceed as described in 8.3.2 and 8.3.3. 9.1.2 Non-available lysine In the case of the manual system, the final reading on the spec- trometer may be
42、made by diluting the volume of the lysine plus reagent mixture to 20 ml with sodium citrate buffer solution of pH 5,28 (5.6) (Let V, be the volume of test solution thus obtained.) 8.5 Number of determinations Carry out two determinations on the same test sample using two different pairs of test port
43、ions. 9 Expression of results 8.1 Method of calculation and formulae 9.1.1 Total lysine 9.1.1.1 Determination using an automatic analyser The total lysine, wl, expressed as a percentage by mass, is equal to 0,365 s, -x- mlxV0 SO where Sc is the area of the peak corresponding to 0,25 pmol of lysine;
44、St is the area of the peak corresponding to the total lysine determined in 8.3.3.1; ml is the mass, in grams, of the test portion placed in the first flask; 9.1.2.1 Determination using an automatic analyser The non-available lysine, 2, expressed as a percentage by mass, is equal to 0,073 s, -x- “2 x
45、 vo so where So is the area of the peak corresponding to 0.25 pmol of lysine; S2 is the area of the peak corresponding to the non- available lysine, determined in 8.4.4; m2 is the mass, in grams, of the test portion placed in the second flask; V. is the volume, in millilitres, of hydrolysate transfe
46、rred to the column (generally, PO = 0,5 ml). 9.1.2.2 Determination using a manual system The non-available lysine, 2, expressed as a percentage by mass, is equal to v, x 0,007 3 A2 x- m2 x vo Ao where A0 is the absorbance of the standard lysine solution deter- mined in 8.3.3.2.3; A2 is the absorbanc
47、e determined in 8.4.4; 4 , Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/27/2007 09:21:27 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 5510-1984 (E) Vc
48、 is the volume, in millilitres, of hydrolysate transferred NOTE - The available lysine, expressed as a percentage by mass of to the column (generally, Vc = 1 ml); total lysine is given by the formula V, is the volume, in millilitres, of the test solution (gener- ally, V2 = 20 ml) ; (Wl -w2) x 100 WI
49、 m2 is the mass, in grams, of the test portion placed in the second flask. 9.1.3 Available lysine 9.2 Repeatability The difference between the results of two determinations carried out simultaneously or in rapid succession by the same analyst shall not exceed 10 % of the mean value of the results obtained. The available lysine, w, expressed as a percentage by mass, is equal to 10 Test report Wl -w2 The test