Commission Regulation (EC) No 273/2008 (repealed)Show full title

7. PROCEDURE

7.1. Preparation of test samples

Use for test sample preparation one of the three following methods of milk fat extraction.

7.1.1. Isolation from butter or butteroil

Melt 50 g to 100 g of test sample at 50 °C using a water bath (5.5) or an oven (5.6). Place 0,5 to 1,0 g of sodium sulfate (4.7) in a folded filter paper (5.12). Preheat a 250 ml Erlenmeyer flask (5.10) and a funnel (5.11) with inserted filter paper in the oven (5.6) set at 50 °C. Filter, while maintaining the preheated flask, funnel and inserted filter device in the oven, the fat layer of the molten sample. Take care that no serum is transferred.

Only in cases where a limited amount of test sample is available, a smaller test sample may be used and the procedure should be adapted accordingly. However, the handling of a smaller test portion involves a higher risk of obtaining a non-representative sample.

Note 1: Butter can be obtained from cream by churning and thorough washing of the resulting butter grain.

Note 2: The milk fat obtained using the procedure in 7.1.1 will be almost free of phospholipids.

7.1.2. Extraction according to the Röse–Gottlieb gravimetric method

Extract the fat fraction from the test sample by using the gravimetric method described in one of the Standards ISO 1211 | IDF 001D, ISO 2450 | IDF 016C or ISO 7328 | IDF 116A.

Note: If phospholipids are present in the milk fat obtained, a cholesterol peak will be obtained which is increased by approximately 0,1 %. The TG composition standardized to 100 % including cholesterol is thereby influenced only to a negligible extent.

7.1.3. Extraction from milk using silica gel columns

Add, using a microlitre pipette (5.7), 0,7 ml of test sample tempered to 20 °C to a 1 ml to 3 ml Extrelut column (5.3). Allow to distribute uniformly on the silica gel for approximately 5 min.

To denature the protein–lipid complexes, add, using the graduated pipette (5.8), 1,5 ml of methanol (4.3) to the Extrelut column. Subsequently, extract the fat fraction from the test sample with 20 ml of n-hexane (4.4). Add the n-hexane slowly in small amounts. Collect the solvent draining off in a 50 ml round-bottomed flask (5.9) previously dried to a constant, known mass weighed to the nearest 1 mg, recording the mass to 0,1 mg.

Allow the column to drain until empty after the extraction. Distil off the solvents from the eluate on a rotary evaporator (5.13) with its water bath set at between 40 °C and 50 °C. After the solvents have been distilled off, dry and subsequently weigh the round-bottomed flask and its contents to the nearest 1 mg, recording the mass to 0,1 mg. Determine the fat mass yield by subtracting the mass of the dried empty round-bottomed flask from the mass obtained.

Note: Fat extractions by the Gerber, Weibull–Berntrop or Schmid–Bondzynski–Ratzlaff methods or isolation of milk fat using detergents (BDI method) are not suitable for TG analysis, because substantial quantities of partial glycerides or phosholipids can pass into the fat phase. Consequently, the application of this International Standard is limited regarding certain products, particularly cheese.

7.2. Preparation of sample solution

For gas chromatography with a packed column, prepare a 5 % (volume fraction) solution of the fat (obtained according to 7.1) in n-hexane (4.4) or n-heptane (4.5). Depending on the column dimensions, use a concentration of 1 % (0,53 mm, ID wide-bore) or lower for on-column injection with a capillary column.

Based on the column used and the mass of fat obtained in 7.1.3, determine the amount of solvent (4.4 or 4.5) to be added to the test sample material in the flask on the basis of weighing to the nearest 1 mg, and recording the mass to 0,1 mg. Completely dissolve the remainder.

Transfer approximately 1 ml of the sample solution into an ampoule (5.14).

7.3. Chromatographic triglyceride determination

7.3.1. Baseline drift

To minimize baseline rising, the column shall be conditioned as specified in 5.2.2 (capillary column) or in Annex A.4 (packed column).

Note: Because of the high column temperature, the analysis of TGs is particularly susceptible to a rise of the baseline in the high carbon-number range.

7.3.2. Injection technique

7.3.2.1. Packed column

To avoid discrimination effects, apply the hot-needle technique for improving the quantification of the high-boiling TG components. Fill the needle with air by drawing up the fat solution in the syringe. Insert the needle into the injector. Heat the needle up prior to injection for about 3 s. Then, rapidly inject the syringe content.

7.3.2.2. Capillary column

When using cold on-column injection (7.3.4.2), insert the needle of the syringe and inject immediately. The dwell time of the needle in the injection port should be such that broad tailing of the solvent peak is avoided.

Note: The optimum dwell time typically is about 3 s.

7.3.3. Calibration

7.3.3.1. General

For the calibration of test samples, perform two to three analyses of standardized milk fat at the beginning of every day. Use the last analysis of the standardized milk fat to determine the response factors, RF_si (mass fraction/area fraction) of the TGs and of cholesterol and apply these to the subsequent test samples (see 9.1):

(1)

where:

w _si

is the mass fraction, expressed as a percentage, of each TG or cholesterol in the standardized milk fat;

A _si

is the numerical value of the peak area of each TG or cholesterol in the standardized milk fat.

Use either 7.3.3.2 or 7.3.3.3 to obtain a standardized milk fat with a known TG composition.

7.3.3.2. Commercial milk fat standard

The best way to determine the response factor of each constituent of the test sample is to use a standardized milk fat with a certified TG composition.

Note: A suitable standard is CRM 519 (anhydrous milk fat) obtainable from the Institute for Reference Materials and Measurements (IRMM), Geel, Belgium(1)).

7.3.3.3. Laboratory milk fat standard

Prepare about 1 g of a mixture of the fat standards (see 4.2, containing at least the saturated TGs, C₂₄, C₃₀, C₃₆, C₄₂, C₄₈ and C₅₄, as well as cholesterol; plus, preferably, C₅₀ and C₅₂) by weighing to the nearest 1 mg, recording the mass to 0,1 mg, to obtain a relative TG composition similar to milk fat.

Analyse repeatedly a solution of the fat standards mixture in n-hexane (4.4) or n-heptane (4.5) according to 7.3.4. In the same sequence, analyse repeatedly averagely composed milk fat.

Determine the TG response factors from the fat standards mixture. Intermediate response factors of TGs not present in the mixture can be calculated by mathematical interpolation. Apply the response factors obtained to the milk fat, in order to obtain a standardized composition. The standardized milk fat thus obtained has a stock life of several years, if stored under nitrogen at a maximum temperature of -18 °C.

7.3.4. Chromatographic conditions

Note: Use of either packed or capillary columns generally results in a resolution similar to Figure 1. Splitting of the even-numbered TGs is not normally observed and shall be avoided.

7.3.4.1. Packed column

(a)

Temperature programme: Set the initial oven temperature at 210 °C. Maintain it at that temperature for 1 min. Then increase the temperature at a rate of 6 °C/min to 350 °C. Maintain it at that (final) temperature for 5 min.

(b)

Detector and injector temperatures: Set both at 370 °C.

(c)

Carrier gas: Use nitrogen at a constant flow rate of about 40 ml/min. Adjust the exact carrier gas flow in such a manner that C₅₄ is eluted at 341 °C.

(d)

Duration of analysis: 29,3 min.

(e)

Injection volume: Inject 0,5 µl of a 5 % (volume fraction) sample solution.

If no TG analyses are carried out, maintain the initial oven temperature as given in a), the detector and injector temperatures as in b) and the carrier gas flow rate as in c) at constant level, also overnight and during weekends and holidays. This ensures best performance of the column.

7.3.4.2. Capillary column

(a)

Temperature programme: Set the initial oven temperature at 80 °C. Maintain it at that temperature for 0,5 min. Then increase the temperature at a rate of 50 °C/min to 190 °C and subsequently at a rate of 6 °C/min to 350 °C. Maintain it at that (final) temperature for 5 min.

(b)

Detector temperature: Set at 370 °C.

(c)

Carrier gas: Use nitrogen at a constant flow rate of about 3 ml/min.

(d)

Duration of analysis: 34,4 min.

(e)

Injection volume: Inject 0,5 µl of a 1 % (volume fraction) sample solution.

Maintain these settings during standby to ensure best performance (see 7.3.4.1).

The analytical settings given in 7.3.4.2 are suitable for use with a wide-bore column (0,53 mm ID) as specified in 5.2.2. Different conditions may be applied if another column dimension or phase is used.

(1)

Example of a suitable product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement of this product.