FIRST COMMISSION DIRECTIVE

of 26 July 1979

Laying down Community methods of analysis for testing certain sugars intended for human consumption

(79/786/EEC)

THE COMMISSION OF THE EUROPEAN COMMUNITIES,

Having regard to the Treaty establishing the European Economic Community,

Having regard to Council Directive 73/437/EEC of 11 December 1973 on the approximation of the laws of the Member States concerning certain sugars intended for human consumption (1), and in particular Article 11 thereof,

Whereas Article 11 of that Directive lays down that the composition of certain sugars shall be verified by Community methods of analysis;

Whereas it is desirable to adopt an initial series of methods in respect of which studies have been completed;

Whereas the method of determining the colour type for sugar or white sugar and for extra-white sugar, the method of measuring the conductivity ash in extra-white sugar, in sugar solution, in invert sugar solution and in invert sugar syrup, and the method of determining the colour in solution of extra-white sugar and sugar solution are laid down in the Annex to Directive 73/437/EEC;

Whereas, on the other hand, pending the formulation of further Community methods for the determination of reducing sugars, it would be advisable to allow the Member States the option of continuing to authorize the use of the Lane and Eynon method (methods 7 and 8 in Annex II, III.3 and III.4) instead of the Luff-Schoorl method (method 6 in Annex 11, III.3 and III.4);

Whereas the methods of analysis provided for in this Directive are in accordance with the opinion of the Standing Committee on Foodstuffs,

HAS ADOPTED THIS DIRECTIVE:

Article 1

1. Member States shall require that the analyses necessary for verification of the criteria set out in Annex I be performed according to the methods described in Annex II to this Directive.

2. Without prejudice to the second subparagraph, the Luff-Schoorl method (Annex II, method 6) shall be used to determine the reducing sugars in the following sugars:

Member States may, however, require the use in their territory of the Lane and Eynon method (Annex II, methods 7 and/or 8 as appropriate) to determine the reducing sugars in one or more of the sugars listed above.

3. If a Member State makes use of the option provided for in the second subparagraph of paragraph 2, it shall forthwith inform the Commission and the other Member States thereof.

Article 2

Member States shall bring into force the laws, regulations or administrative provisions necessary to comply with this Directive not later than 18 months following its notification. They shall forthwith inform the Commission thereof.

Article 3

This Directive is addressed to the Member States.

(1) OJ No L 356, 27. 12. 1973, p. 71.

Determination of the loss of mass on drying in:

SCOPE OF TOE COMMUNITY METHODS OF ANALYSIS FOR CERTAIN SUGARS INTENDED FOR HUMAN CONSUMPTION

ANNEX II

METHODS OF ANALYSIS TO VERIFY THE COMPOSITION OF CERTAIN SUGARS INTENDED FOR HUMAN CONSUMPTION

INTRODUCTION

1. Preparation of the sample for analysis

Thoroughly mix the sample received at the laboratory.

Remove a sub-sample of at least 200 g and transfer immediately to a clean, dry, moisture-tight vessel fitted with an airtight closure.

2. Reagents and apparatus

In the description of the apparatus, reference is made only to special instruments and apparatus or to those calling for special standards.

Wherever mention is made of water, this means distilled water or demineralized water of at least equivalent purity.

All reagents shall be of analytical reagent quality unless otherwise specified.

Wherever reference is made to a reagent solution without further qualification, an aqueous solution is meant.

3. Expression of results

The result referred to in the official analysis report shall be the mean value of at least two satisfactory replicate determinations.

Unless otherwise stated the results shall be expressed as a percentage by mass of the original sample as received at the laboratory.

The number of significant figures in the result so expressed shall be governed by the precision of the method.

METHOD 1

DETERMINATION OF THE LOSS OF MASS ON DRYING

1. Scope and field of application

The method determines the loss of mass on drying in:

— semi-white sugar,

— sugar or white sugar,

— extra-white sugar.

2. Definition

‘Loss of mass on drying’: the value of the loss of mass on drying as determined by the method specified.

3. Principle

The loss of mass on drying is determined by drying at a temperature of 103 ± 2 oC.

4. Apparatus

4.1. Analytical balance, accurate to within 0.1 mg.

4.2. Oven, suitably ventilated, thermostatically controlled, and capable of being maintained at 103 ± 2 oC.

4.3. Metal weighing dish, flat-bottomed, resistant to attack by the samples and the conditions of test, diameter at least 100 mm, depth at least 30 mm.

4.4. Desiccator, containing freshly activated silica gel or an equivalent desiccant, with a water content indicator.

5. Procedure

N.B.: The operations described in sections 5.3 to 5.7 must be performed immediately after opening the sample container.

5.1. Dry the dish (4.3) to constant weight in the oven (4.2) at 103 ± 2 oC.

5.2. Allow the dish to cool in the desiccator (4.4) for at least 30 to 35 minutes and then weigh to the nearest 0.l mg.

5.3. Weigh accurately, to the nearest 0.1 mg, approximately 20 to 30 g of the sample into the dish.

5.4. Place the dish in the oven (4.2) at 103 ± 2 oC for three hours.

5.5. Allow the dish to cool in a desiccator (4.4) and weigh to the nearest 0.1 mg.

5.6. Replace the dish in the oven at 103 ± 2 oC for 30 minutes. Allow to cool in the desiccator (4.4) and weigh to the nearest 01 mg. Repeat this operation if the difference between two weighings is more than 1 mg. Should an increase in mass occur, the lowest recorded reading will be used in the calculation.

5.7. Do not exceed four hours total drying time.

6. Expression of results

6.1. Formula and method of calculation

The loss of mass on drying, as a percentage by mass of the sample, is given by the following formula:

where:

m0 is the initial mass, in grams, of the test portion, m1 is the mass, in grams, of the test portion after drying.

6.2. Repeatability

The difference between the results of two determinations when carried out simultaneously or in rapid succession on the same sample, by the same analyst, under the same conditions, shall not exceed 0·02 g per 100 g of sample.

METHOD 2

DETERMINATION OF DRY MATTER

Vacuum oven method

1. Scope and field of application

The method determines the dry matter content in:

— glucose syrup,

— dried glucose syrup,

— dextrose monohydrate,

— dextrose anhydrous.

2. Definition

‘The dry matter content’: the content of dry matter as determined by the method specified.

3. Principle

The dry matter is determined at a temperature of 70 ± 1 oC using a vacuum oven at a pressure not exceeding 3·3 kPa (34 mbar). The test portions in the case of glucose syrup or dried glucose syrups, are prepared by mixing with water and kieselguhr before drying.

4. Reagents

4.1. Kieselguhr: place in a Buchner funnel and purify by repeated washings with dilute hydrochloric acid (1 ml of concentrated acid, density at 20 oC = 1·19 g/ml per litre of water). The treatment is complete when the washings remain definitely acid. Wash with water until the pH value of the filtered water is greater than 4. Dry in an oven at 103 ± 2 oC and store in an airtight container.

5. Apparatus

5.1. Vacuum drying oven, leak tight, thermostatically controlled and equipped with a thermometer and a vacuum manometer. The oven design must be such that the heat is rapidly transferred to the weighing dishes placed on the shelves.

5.2. Air-drying train consisting of a glass tower filled with freshly activated dry silica gel or an equivalent desiccant containing a water content indicator. This tower is mounted in series with a gas scrubber containing concentrated sulphuric acid connected to the air intake of the oven.

5.3. Vaccum pump capable of maintaining the presure in the oven at 3·3 kPa (34 mbar) or less.

5.4. Metal weighing dish, flat-bottomed, resistant to attack by the samples and the conditions of test, diameter at least 100 mm, depth at least 300 mm.

5.5. Glass rod of a length such that it cannot completely fall into the container.

5.6. Desiccator containing freshly activated dry silica gel, or an equivalent desiccant, with a water content indicator.

5.7. Analytical balance accurate to within 0.1 mg.

6. Procedure

6.1. Pour approximately 30 g of kieselguhr (4.1) into the weighing dish (5.4) equipped with a glass rod (5.5). Place the whole in the oven (5.1) at 70 ± 1 oC and reduce the pressure to 3·3 kPa (34 mbar) or less. Dry for at least five hours, drawing a slow stream of air into the oven through the drying train. Check the pressure from time to time and correct it if necessary.

6.2. Restore atmospheric pressure in the oven by cautiously increasing the intake of dry air. Immediately place the dish together with the glass rod in the desiccator (5.6). Allow to cool and then weigh.

63. Accurately weigh to the nearest 1 mg approximately 10 g of the sample to be analyzed into a 100 ml beaker.

6.4. Dilute the test portion with 10 ml of warm water and transfer the solution quantitatively into the weighing dish, using the glass rod (5.5).

6.5. Place the dish containing the test portion and the glass rod in the oven and reduce the pressure to 3.3 kPa (34 mbar) or less. Dry at 70 ± 1 oC, allowing a slow stream of dry air to pass through the oven. The drying operation should proceed for 20 hours; the bulk of the loss should occur towards the end of the first day. It will be necessary to keep the vacuum pump working at a preset pressure and allow a slow stream of dry air to enter the oven so as to maintain a pressure of approximately 3·3 kPa (34 mbar) or less during the night.

6.6. Restore atmospheric pressure in the oven by cautiously increasing the intake of dry air. Immediately place the weighing dish and contents in the desiccator. Allow to cool and then weigh to the nearest 1 mg.

6.7. Continue operation (6.5) for a further four hours. Restore atmospheric pressure in the oven and immediately place the dish in the desiccator. Allow to cool and then weigh. Ascertain whether constant mass has been reached. It is considered that constant mass has been satisfactorily attained if the difference between the two weighings of the same dish does not exceed 2 mg. If the difference is greater, repeat operation 6.7.

6.8. For the determination of the dry matter in dextrose anhydrous or dextrose monohydrate samples the use of kieselguhr and water is not required.

7. Expression of results

7.1. Formula and method of calculation

The dry matter content, expressed as a percentage by mass of the sample is given by:

where:

m0 = the initial mass, in grams, of the test portion, m1 = the mass, in grams, of the weighing dish plus the kieselguhr, the glass rod and the residue of the test portion after drying, m2 = the mass, in grams, of the weighing dish plus the kieselguhr and the glass rod.

7.2. Repeatability

The difference between the results of two determinations when carried out simultaneously or in rapid succession on the same sample, by the same analyst, under the same conditions, shall not exceed 0·12 g per 100 g of sample.

METHOD 3

DETERMINATION OF TOTAL DRY MATTER

(Refractometric method)

1. Scope and field of application

The method determines the dry-matter content in:

— sugar solution,

— white sugar solution,

— invert sugar solution,

— white invert sugar solution,

— invert sugar syrup,

— white invert sugar syrup.

2. Definition

‘Dry matter content’: the content of dry matter as determined by the method specified.

3. Principle

The refractive index of a test portion is determined at 20 oC and converted into dry matter content by reference to tables showing the concentration as a function of the refractive index.

4. Apparatus

4.1. Refractometer, accurate to four decimal places, provided with a thermometer and a water-circulation pump connected to a water-bath thermostatically controlled at 20 ± 0·5 oC.

4.2. Light source consisting of a sodium vapour lamp.

5. Procedure

5.1. If any crystals are present in the sample, redissolve them by diluting the sample in the ratio 1 : 1 (m/m).

5.2. Measure the refractive index of the sample at 20 oC in the refractometer (4.1).

6. Expression and calculation of results

6.1. Calculate the dry matter content from the refractive indices for sucrose solutions at 20 oC in the table given and correct for the presence of invert sugars by adding to the result obtained from the tables, 0.022 for every 1 % of invert sugar present in the sample as analyzed.

6.2. If the sample was diluted to 1: 1 (m/m) with water, the calculated dry matter content must be multiplied by two.

6.3. Repeatability The difference between the results of two determinations when carried out simultaneously or in rapid succession on the same sample, by the same analyst, under the same conditions, shall not exceed 0·2 g dry matter per 100 g of sample.

REFERENCE TABLES

Refractive indices (n) of sucrose solutions at 20 oC (1)

n (20 oC) Sucrose (%) 1·3330 0·009 1·3331 0·078 1·3332 0·149 1·3333 0·218 1·3334 0·288 1·3335 0·358 1·3336 0·428 1·3337 0·498 1·3338 0·567 1·3339 0·637 1·3340 0·707 1·3341 0·776 1·3342 0·846 1·3343 0·915 1·3344 0·985 1·3345 1·054 1·3346 1·124 1·3347 1·193 1·3348 1·263 1·3349 1·332 1·3350 1·401 1·3351 1·470 1·3352 1·540 1·3353 1·609 1·3354 1·678 1·3355 1·747 1·3356 1·816 1·3357 1·885 1·3358 1·954 1·3359 2·023 1·3360 2·092 1·3361 2·161 1·3362 2·230 1·3363 2·299 1·3364 2·367 1·3365 2·436 1·3366 2·505 1·3367 2·574 1·3368 2·642 1·3369 2·711 1·3370 2·779 1·3371 2·848 1·3372 2·917 1·3373 2·985 1·3374 3·053 1·3375 3·122 1·3376 3·190 1·3377 3·259 1·3378 3·327 1·3379 3·395 1·3380 3·463 1·3381 3·532 1·3382 3·600 1·3383 3·668 1·3384 3·736 1·3385 3·804 1·3386 3·872 1·3387 3·940 1·3388 4·008 1·3389 4·076 1·3390 4·144 1·3391 4·212 1·3392 4·279 1·3393 4·347 1·3394 4·415 1·3395 4·483 1·3396 4·550 1·3397 4·618 1·3398 4·686 1·3399 4·753 1·3400 4·821 1·3401 4·888 1·3402 4·956 1·3403 5·023 1·3404 5·091 1·3405 5·158 1·3406 5·225 1·3407 5·293 1·3408 5·360 1·3409 5·427 1·3410 5·494 1·3411 5·562 1·3412 5·629 1·3413 5·696 1·3414 5·763 1·3415 5·830 1·3416 5·897 1·3417 5·964 1·3418 6·031 1·3419 6·098 1·3420 6·165 1·3421 6·231 1·3422 6·298 1·3423 6·365 1·3424 6·432 1·3425 6·498 1·3426 6·565 1·3427 6·632 1·3428 6·698 1·3429 6·765 1·3430 6·831 1·3431 6·898 1·3432 6·964 1·3433 7·031 1·3434 7·097 1·3435 7·1642 1·3436 7·230 1·3437 7·296 1·3438 7·362 1·3439 7·429 1·3440 7·495 1·3441 7·561 1·3442 7·627 1·3443 7·693 1·3444 7·759 1·3445 7·825 1·3446 7·891 1·3447 7·957 1·3448 8·023 1·3449 8·089 1·3450 8·155 1·3451 8·221 1·3452 8·287 1·3453 8·352 1·3454 8·418 1·3455 8·484 1·3456 8·550 1·3457 8·615 1·3458 8·681 1·3459 8·746 1·3460 8·812 1·3461 8·878 1·3462 8·943 1·3463 9·008 1·3464 9·074 1·3465 9·139 1·3466 9·205 1·3467 9·270 1·3468 9·335 1·3469 9·400 1·3470 9·466 1·3471 9·531 1·3472 9·596 1·3473 9·661 1·3474 9·726 1·3475 9·791 1·3476 9·856 1·3477 9·921 1·3478 9·986 1·3479 10·051 1·3480 10·116 1·3481 10·181 1·3482 10·246 1·3483 10·311 1·3484 10·375 1·3485 10·440 1·3486 10·505 1·3487 10·570 1·3488 10·634 1·3489 10·699 1·3490 10·763 1·3491 10·828 1·3492 10·892 1·3493 10·957 1·3494 11·021 1·3495 11·086 1·3496 11·150 1·3497 11·215 1·3498 11·279 1·3499 11·343 1·3500 11·407 1·3501 11·472 1·3502 11·536 1·3503 11·600 1·3504 11·664 1·3505 11·728 1·3506 11·792 1·3507 11·856 1·3508 11·920 1·3509 11·984 1·3510 12·048 1·3511 12·112 1·3512 12·176 1·3513 12·240 1·3514 12·304 1·3515 12·368 1·3516 12·431 1·3517 12·495 1·3518 12·559 1·3519 12·623 1·3520 12·686 1·3521 12·750 1·3522 12·813 1·3523 12·877 1·3524 12·940 1·3525 13·004 1·3526 13·067 1·3527 13·131 1·3528 13·194 1·3529 13·258 1·3530 13·321 1·3531 13·384 1·3532 13·448 1·3533 13·511 1·3534 13·574 1·3535 13·637 1·3536 13·700 1·3537 13·763 1·3538 13·826 1·3539 13·890 1·3540 13·953 1·3541 14·016 1·3542 14·079 1·3543 14·141 1·3544 14·204 1·3545 14·267 1·3546 14·330 1·3547 14·393 1·3548 14·456 1·3549 14·518 1·3550 14·581 1·3551 14·644 1·3552 14·707 1·3553 14·769 1·3554 14·832 1·3555 14·894 1·3556 14·957 1·3557 15·019 1·3558 15·082 1·3559 15·144 1·3560 15·207 1·3561 15·269 1·3562 15·332 1·3563 15·394 1·3564 15·456 1·3565 15·518 1·3566 15·581 1·3567 15·643 1·3568 15·705 1·3569 15·767 1·3570 15·829 1·3571 15·891 1·3572 15·953 1·3573 16016 1·3574 16·078 1·3575 16·140 1·3576 16·201 1·3577 16·263 1·3578 16·325 1·3579 16·387 1·3580 16·449 1·3581 16·511 1·3582 16·573 1·3583 16·634 1·3584 16·696 1·3585 16·758 1·3586 16·819 1·3587 16·881 1·3588 16·943 1·3589 17·004 1·3590 17·066 1·3591 17·127 1·3592 17·189 1·3593 17·250 1·3594 17·311 1·3595 17·373 1·3596 17·434 1·3597 17·496 1·3598 17·557 1·3599 17·618 1·3600 17·679 1·3601 17·741 1·3602 17·802 1·3603 17·863 1·3604 17·924 1·3605 17·985 1·3606 18·046 1·3607 18·107 1·3608 18·168 1·3609 18·229 1·3610 18·290 1·3611 18·351 1·3612 18·412 1·3613 18·473 1·3614 18·534 1·3615 18·595 1·3616 18·655 1·3617 18·716 1·3618 18·777 1·3619 18·837 1·3620 18·898 1·3621 18·959 1·3622 19·019 1·3623 19·080 1·3624 19·141 1·3625 19·201 1·3626 19·262 1·3627 19·322 1·3628 19·382 1·3629 19·443 1·3630 19·503 1·3631 19·564 1·3632 19·624 1·3633 19·684 1·3634 19·745 1·3635 19·805 1·3636 19·865 1·3637 19·925 1·3638 19·985 1·3639 20·045 1·3640 20·106 1·3641 20·166 1·3642 20·226 1·3643 20·286 1·3644 20·346 1·3645 20·406 1·3646 20·466 1·3647 20·525 1·3648 20·585 1·3649 20·645 1·3650 20·705 1·3651 20·765 1·3652 20·825 1·3653 20·884 1·3654 20·944 1·3655 21·004 1·3656 21·063 1·3657 21·123 1·3658 21·183 1·3659 21·242 1·3660 21·302 1·3661 21·361 1·3662 21·421 1·3663 21·480 1·3664 21·540 1·3665 21·599 1·3666 21·658 1·3667 21·718 1·3668 21·777 1·3669 21·836 1·3670 21·896 1·3671 21·955 1·3672 22·014 1·3673 22·073 1·3674 22·132 1·3675 22·192 1·3676 22·251 1·3677 22·310 1·3678 22·369 1·3679 22·428 1·3680 22·487 1·3681 22·546 1·3682 22·605 1·3683 22·664 1·3684 22·723 1·3685 22·781 1·3686 22·840 1·3687 22·899 1·3688 22·958 1·3689 23·017 1·3690 23·075 1·3691 23·134 1·3692 23·193 1·3693 23·251 1·3694 23·310 1·3695 23·369 1·3696 23·427 1·3697 23·486 1·3698 23·544 1·3699 23·603 1·3700 23·661 1·3701 23·720 1·3702 23·778 1·3703 23·836 1·3704 23·895 1·3705 23·953 1·3706 24·011 1·3707 24·070 1·3708 24·128 1·3709 24·186 1·3710 24·244 1·3711 24·302 1·3712 24·361 1·3713 24·419 1·3714 24·477 1·3715 24·535 1·3716 24·593 1·3717 24·651 1·3718 24·709 1·3719 24·767 1·3720 24·825 1·3721 24·883 1·3722 24·941 1·3723 24·998 1·3724 25·056 1·3725 25·114 1·3726 25·172 1·3727 25·230 1·3728 25·287 1·3729 25·345 1·3730 25·403 1·3731 25·460 1·3732 25·518 1·3733 25·576 1·3734 25·633 1·3735 25·691 1·3736 25·748 1·3737 25·806 1·3738 25·863 1·3739 25·921 1·3740 25·978 1·3741 26·035 1·3742 26·093 1·3743 26·150 1·3744 26·207 1·3745 26·265 1·3746 26·322 1·3747 26·379 1·3748 26·436 1·3749 26·493 1·3750 26·551 1·3751 26·608 1·3752 26·665 1·3753 26·722 1·3754 26·779 1·3755 26·836 1·3756 26·893 1·3757 26·950 1·3758 27·007 1·3759 27·064 1·3760 27·121 1·3761 27·178 1·3762 27·234 1·3763 27·291 1·3764 27·348 1·3765 27·405 1·3766 27·462 1·3767 27·518 1·3768 27·575 1·3769 27·632 1·3770 27·688 1·3771 27·745 1·3772 27·802 1·3773 27·858 1·3774 27·915 1·3775 27·971 1·3776 28·028 1·3777 28·084 1·3778 28·141 1·3779 28·197 1·3780 28·253 1·3781 28·310 1·3782 28·366 1·3783 28·422 1·3784 28·479 1·3785 28·535 1·3786 28·591 1·3787 28·648 1·3788 28·704 1·3789 28·760 1·3790 28·816 1·3791 28·872 1·3792 28·928 1·3793 28·984 1·3794 29·040 1·3795 29·096 1·3796 29·152 1·3797 29·208 1·3798 29·264 1·3799 29·320 1·3800 29·376 1·3801 29·432 1·3802 29·488 1·3803 29·544 1·3804 29·600 1·3805 29·655 1·3806 29·711 1·3807 29·767 1·3808 29·823 1·3809 29·878 1·3810 29·934 1·3811 29·989 1·3812 30·045 1·3813 30·101 1·3814 30·156 1·3815 30·212 1·3816 30·267 1·3817 30·323 1·3818 30·378 1·3819 30·434 1·3820 30·489 1·3821 30·544 1·3822 30·600 1·3823 30·655 1·3824 30·711 1·3825 30·766 1·3826 30·821 1·3827 30·876 1·3828 30·932 1·3829 30·987 1·3830 31·042 1·3831 31·097 1·3832 31·152 1·3833 31·207 1·3834 31·262 1·3835 31·317 1·3836 31·372 1·3837 31·428 1·3838 31·482 1·3839 31·537 1·3840 31·592 1·3841 31·647 1·3842 31·702 1·3843 31·757 1·3844 31·812 1·3845 31·867 1·3846 31·922 1·3847 31·976 1·3848 32·031 1·3849 32·086 1·3850 32·140 1·3851 32·195 1·3852 32·250 1·3853 32·304 1·3854 32·359 1·3855 32·414 1·3856 32·468 1·3857 32·523 1·3858 32·577 1·3859 32·632 1·3860 32·686 1·3861 32·741 1·3862 32·795 1·3863 32·849 1·3864 32·904 1·3865 32·958 1·3866 33·013 1·3867 33·067 1·3868 33·121 1·3869 33·175 1·3870 33·230 1·3871 33·284 1·3872 33·338 1·3873 33·392 1·3874 33·446 1·3875 33·500 1·3876 33·555 1·3877 33·609 1·3878 33·663 1·3879 33·717 1·3880 33·771 1·3881 33·825 1·3882 33·879 1·3883 33·933 1·3884 33·987 1·3885 34·040 1·3886 34·094 1·3887 34·148 1·3888 34·202 1·3889 34·256 1·3890 34·310 1·3891 34·363 1·3892 34·417 1·3893 34·471 1·3894 34·524 1·3895 34·578 1·3896 34·632 1·3897 34·685 1·3898 34·739 1·3899 34·793 1·3900 34·846 1·3901 34·900 1·3902 34·953 1·3903 35·007 1·3904 35·060 1·3905 35·114 1·3906 35·167 1·3907 35·220 1·3908 35·274 1·3909 35·327 1·3910 35·380 1·3911 35·434 1·3912 35·487 1·3913 35·540 1·3914 35·593 1·3915 35·647 1·3916 35·700 1·3917 35·753 1·3918 35·806 1·3919 35·859 1·3920 35·912 1·3921 35·966 1·3922 36·019 1·3923 36·072 1·3924 36·125 1·3925 36·178 1·3926 36·231 1·3927 36·284 1·3928 36·337 1·3929 36·389 1·3930 36·442 1·3931 36·495 1·3932 36·548 1·3933 36·601 1·3934 36·654 1·3935 36·706 1·3936 36·759 1·3937 36·812 1·3938 36·865 1·3939 36·917 1·3940 36·970 1·3941 37·023 1·3942 37·075 1·3943 37·128 1·3944 37·180 1·3945 37·233 1·3946 37·286 1·3947 37·338 1·3948 37·391 1·3949 37·443 1·3950 37·495 1·3951 37·548 1·3952 37·600 1·3953 37·653 1·3954 37·705 1·3955 37·757 1·3956 37·810 1·3957 37·862 1·3958 37·914 1·3959 37·967 1·3960 38·019 1·3961 38·071 1·3962 38·123 1·3963 38·175 1·3964 38·228 1·3965 38·280 1·3966 38·332 1·3967 38·384 1·3968 38·436 1·3969 38·488 1·3970 38·540 1·3971 38·592 1·3972 38·644 1·3973 38·696 1·3974 38·748 1·3975 38·800 1·3976 38·852 1·3977 38·904 1·3978 38·955 1·3979 39·007 1·3980 39·059 1·3981 39·111 1·3982 39·163 1·3983 39·214 1·3984 39·266 1·3985 39·318 1·3986 39·370 1·3987 39·421 1·3988 39·473 1·3989 39·525 1·3990 39·576 1·3991 39·628 1·3992 39·679 1·3993 39·731 1·3994 39·782 1·3995 39·834 1·3996 39·885 1·3997 39·937 1·3998 39·988 1·3999 40·040 1·4000 40·091 1·4001 40·142 1·4002 40·194 1·4003 40·245 1·4004 40·296 1·4005 40·348 1·4006 40·399 1·4007 40·450 1·4008 40·501 1·4009 40·553 1·4010 40·604 1·4011 40·655 1·4012 40·706 1·4013 40·757 1·4014 40·808 1·4015 40·860 1·4016 40·911 1·4017 40·962 1·4018 41·013 1·4019 41·064 1·4020 41·115 1·4021 41·166 1·4022 41·217 1·4023 41·268 1·4024 41·318 1·4025 41·369 1·4026 41·420 1·4027 41·471 1·4028 41·522 1·4029 41·573 1·4030 41·623 1·4031 41·674 1·4032 41·725 1·4033 41·776 1·4034 41·826 1·4035 41·877 1·4036 41·928 1·4037 41·978 1·4038 42·029 1·4039 42·080 1·4040 42·130 1·4041 42·181 1·4042 42·231 1·4043 42·282 1·4044 42·332 1·4045 42·383 1·4046 42·433 1·4047 42·484 1·4048 42·534 1·4049 42·585 1·4050 42·635 1·4051 42·685 1·4052 42·736 1·4053 42·786 1·4054 42·836 1·4055 42·887 1·4056 42·937 1·4057 42·987 1·4058 43·037 1·4059 43·088 1·4060 43·138 1·4061 43·188 1·4062 43·238 1·4063 43·288 1·4064 43·338 1·4065 43·388 1·4066 43·439 1·4067 43·489 1·4068 43·539 1·4069 43·589 1·4070 43·639 1·4071 43·689 1·4072 43·739 1·4073 43·789 1·4074 43·838 1·4075 43·888 1·4076 43·938 1·4077 43·988 1·4078 44·038 1·4079 44·088 1·4080 44·138 1·4081 44·187 1·4082 44·237 1·4083 44·287 1·4084 44·337 1·4085 44·386 1·4086 44·436 1·4087 44·486 1·4088 44·535 1·4089 44·585 1·4090 44·635 1·4091 44·684 1·4092 44·734 1·4093 44·783 1·4094 44·833 1·4095 44·882 1·4096 44·932 1·4097 44·981 1·4098 45·031 1·4099 45·080 1·4100 45·130 1·4101 45·179 1·4102 45·228 1·4103 45·278 1·4104 45·327 1·4105 45·376 1·4106 45·426 1·4107 45·475 1·4108 45·524 1·4109 45·574 1·4110 45·623 1·4111 45·672 1·4112 45·721 1·4113 45·770 1·4114 45·820 1·4115 45·869 1·4116 45·918 1·4117 46·967 1·4118 46·016 1·4119 46·065 1·4120 46·114 1·4121 46·163 1·4122 46·212 1·4123 46·261 1·4124 46·310 1·4125 46·359 1·4126 46·408 1·4127 46·457 1·4128 46·506 1·4129 46·555 1·4130 46·604 1·4131 46·652 1·4132 46·701 1·4133 46·750 1·4134 46·799 1·4135 46·848 1·4136 46·896 1·4137 46·945 1·4138 46·994 1·4139 47·043 1·4140 47·091 1·4141 47·140 1·4142 47·188 1·4143 47·237 1·4144 47·286 1·4145 47·334 1·4146 47·383 1·4147 47·431 1·4148 47·480 1·4149 47·528 1·4150 47·577 1·4151 47·625 1·4152 47·674 1·4153 47·722 1·4154 47·771 1·4155 47·819 1·4156 47·868 1·4157 47·916 1·4158 47·964 1·4159 48·013 1·4160 48·061 1·4161 48·109 1·4162 48·158 1·4163 48·206 1·4164 48·254 1·4165 48·302 1·4166 48·350 1·4167 48·399 1·4168 48·447 1·4169 48·495 1·4170 48·543 1·4171 48·591 1·4172 48·639 1·4173 48·687 1·4174 48·735 1·4175 48·784 1·4176 48·832 1·4177 48·880 1·4178 48·928 1·4179 48·976 1·4180 49·023 1·4181 49·071 1·4182 49·119 1·4183 49·167 1·4184 49·215 1·4185 49·263 1·4186 49·311 1·4187 49·359 1·4188 49·407 1·4189 49·454 1·4190 49·502 1·4191 49·550 1·4192 49·598 1·4193 49·645 1·4194 49·693 1·4195 49·741 1·4196 49·788 1·4197 49·836 1·4198 49·884 1·4199 49·931 1·4200 49·979 1·4201 50·027 1·4202 50·074 1·4203 50·122 1·4204 50·169 1·4205 50·217 1·4206 50·264 1·4207 50·312 1·4208 50·359 1·4209 50·407 1·4210 50·454 1·4211 50·502 1·4212 50·549 1·4213 50·596 1·4214 50·644 1·4215 50·691 1·4216 50·738 1·4217 50·786 1·4218 50·833 1·4219 50·880 1·4220 50·928 1·4221 50·975 1·4222 51·022 1·4223 51·069 1·4224 51·116 1·4225 51·164 1·4226 51·211 1·4227 51·258 1·4228 51·305 1·4229 51·352 1·4230 51·399 1·4231 51·446 1·4232 51·493 1·4233 51·540 1·4234 51·587 1·4235 51·634 1·4236 51·681 1·4237 51·728 1·4238 51·775 1·4239 51·822 1·4240 51·869 1·4241 51·916 1·4242 51·963 1·4243 52·010 1·4244 52·057 1·4245 52·104 1·4246 52·150 1·4247 52·197 1·4248 52·244 1·4249 52·291 1·4250 52·338 1·4251 52·384 1·4252 52·431 1·4253 52·478 1·4254 52·524 1·4255 52·571 1·4256 52·618 1·4257 52·664 1·4258 52·711 1·4259 52·758 1·4260 52·804 1·4261 52·851 1·4262 52·897 1·4263 52·944 1·4264 52·990 1·4265 53·037 1·4266 53·083 1·4267 53·130 1·4268 53·176 1·4269 53·223 1·4270 53·269 1·4271 53·316 1·4272 53·362 1·4273 53·408 1·4274 53·455 1·4275 53·501 1·4276 53·548 1·4277 53·594 1·4278 53·640 1·4279 53·686 1·4280 53·733 1·4281 53·779 1·4282 53·825 1·4283 53·871 1·4284 53·918 1·4285 53·964 1·4286 54·010 1·4287 54·056 1·4288 54·102 1·4289 54·148 1·4290 54·194 1·4291 54·241 1·4292 54·287 1·4293 54·333 1·4294 54·379 1·4295 54·425 1·4296 54·471 1·4297 54·517 1·4298 54·563 1·4299 54·609 1·4300 54·655 1·4301 54·701 1·4302 54·746 1·4303 54·792 1·4304 54·838 1·4305 54·884 1·4306 54·930 1·4307 54·976 1·4308 55·022 1·4309 55·067 1·4310 55·113 1·4311 55·159 1·4312 55·205 1·4313 55·250 1·4314 55·296 1·4315 55·342 1·4316 55·388 1·4317 55·433 1·4318 55·479 1·4319 55·524 1·4320 55·570 1·4321 55·616 1·4322 55·661 1·4323 55·707 1·4324 55·752 1·4325 55·798 1·4326 55·844 1·4327 55·889 1·4328 55·935 1·4329 55·980 1·4330 56·026 1·4331 56·071 1·4332 56·116 1·4333 56·162 1·4334 56·207 1·4335 56·253 1·4336 56·298 1·4337 56·343 1·4338 56·389 1·4339 56·434 1·4340 56·479 1·4341 56·525 1·4342 56·570 1·4343 56·615 1·4344 56·660 1·4345 56·706 1·4346 56·751 1·4347 56·796 1·4348 56·841 1·4349 56·887 1·4350 56·932 1·4351 56·977 1·4352 57·022 1·4353 57·067 1·4354 57·112 1·4355 57·157 1·4356 57·202 1·4357 57·247 1·4358 57·292 1·4359 57·337 1·4360 57·382 1·4361 57·427 1·4362 57·472 1·4363 57·517 1·4364 57·562 1·4365 57·607 1·4366 57·652 1·4367 57·697 1·4368 57·742 1·4369 57·787 1·4370 57·832 1·4371 57·877 1·4372 57·921 1·4373 57·966 1·4374 58·011 1·4375 58·056 1·4376 58·101 1·4377 58·145 1·4378 58·190 1·4379 58·235 1·4380 58·279 1·4381 58·324 1·4382 58·369 1·4383 58·413 1·4384 58·458 1·4385 58·503 1·4386 58·547 1·4387 58·592 1·4388 58·637 1·4389 58·681 1·4390 58·726 1·4391 58·770 1·4392 58·815 1·4393 58·859 1·4394 58·904 1·4395 58·948 1·4396 58·993 1·4397 59·037 1·4398 59·082 1·4399 59·126 1·4400 59·170 1·4401 59·215 1·4402 59·259 1·4403 59·304 1·4404 59·348 1·4405 59·392 1·4406 59·437 1·4407 59·481 1·4408 59·525 1·4409 59·569 1·4410 59·614 1·4411 59·658 1·4412 59·702 1·4413 59·746 1·4414 59·791 1·4415 59·835 1·4416 59·879 1·4417 59·923 1·4418 59·967 1·4419 60·011 1·4420 60·056 1·4421 60·100 1·4422 60·144 1·4423 60·188 1·4424 60·232 1·4425 60·276 1·4426 60·320 1·4427 60·364 1·4428 60·408 1·4429 60·452 1·4430 60·496 1·4431 60·540 1·4432 60·584 1·4433 60·628 1·4434 60·672 1·4435 60·716 1·4436 60·759 1·4437 60·803 1·4438 60·847 1·4439 60·891 1·4440 60·935 1·4441 60·979 1·4442 61·023 1·4443 61·066 1·4444 61·110 1·4445 61·154 1·4446 61·198 1·4447 61·241 1·4448 61·285 1·4449 61·329 1·4450 61·372 1·4451 61·416 1·4452 61·460 1·4453 61·503 1·4454 61·547 1·4455 61·591 1·4456 61·634 1·4457 61·678 1·4458 61·721 1·4459 61·765 1·4460 61·809 1·4461 61·852 1·4462 61·896 1·4463 61·939 1·4464 61·983 1·4465 62·026 1·4466 62·070 1·4467 62·113 1·4468 62·156 1·4469 62·200 1·4470 62·243 1·4471 62·287 1·4472 62·330 1·4473 62·373 1·4474 62·417 1·4475 62·460 1·4476 62·503 1·4477 62·547 1·4478 62·590 1·4479 62·633 1·4480 62·677 1·4481 62·720 1·4482 62·763 1·4483 62·806 1·4484 62·849 1·4485 62·893 1·4486 62·936 1·4487 62·979 1·4488 63·022 1·4489 63·065 1·4490 63·108 1·4491 63·152 1·4492 63·195 1·4493 63·238 1·4494 63·281 1·4495 63·324 1·4496 63·367 1·4497 63·410 1·4498 63·453 1·4499 63·496 1·4500 63·539 1·4501 63·582 1·4502 63·625 1·4503 63·668 1·4504 63·711 1·4505 63·754 1·4506 63·797 1·4507 63·840 1·4508 63·882 1·4509 63·925 1·4510 63·968 1·4511 64·011 1·4512 64·054 1·4513 64·097 1·4514 64·139 1·4515 64·182 1·4516 64·225 1·4517 64·268 1·4518 64·311 1·4519 64·353 1·4520 64·396 1·4521 64·439 1·4522 64·481 1·4523 64·524 1·4524 64·567 1·4525 64·609 1·4526 64·652 1·4527 64·695 1·4528 64·737 1·4529 64·780 1·4530 64·823 1·4531 64·865 1·4532 64·908 1·4533 64·950 1·4534 64·993 1·4535 65·035 1·4536 65·078 1·4537 65·120 1·4538 65·163 1·4539 65·205 1·4540 65·248 1·4541 65·290 1·4542 65·333 1·4543 65·375 1·4544 65·417 1·4545 65·460 1·4546 65·502 1·4547 65·544 1·4548 65·587 1·4549 65·629 1·4550 65·672 1·4551 65·714 1·4552 65·756 1·4553 65·798 1·4554 65·841 1·4555 65·883 1·4556 65·925 1·4557 65·967 1·4558 66·010 1·4559 66·052 1·4560 66·094 1·4561 66·136 1·4562 66·178 1·4563 66·221 1·4564 66·263 1·4565 66·305 1·4566 66·347 1·4567 66·389 1·4568 66·431 1·4569 66·473 1·4570 66·515 1·4571 66·557 1·4572 66·599 1·4573 66·641 1·4574 66·683 1·4575 66·725 1·4576 66·767 1·4577 66·809 1·4578 66·851 1·4579 66·893 1·4580 66·935 1·4581 66·977 1·4582 67·019 1·4583 67·061 1·4584 67·103 1·4585 67·145 1·4586 67·186 1·4587 67·228 1·4588 67·270 1·4589 67·312 1·4590 67·354 1·4591 67·396 1·4592 67·437 1·4593 67·479 1·4594 67·521 1·4595 67·563 1·4596 67·604 1·4597 67·646 1·4598 67·688 1·4599 67·729 1·4600 67·771 1·4601 67·813 1·4602 67·854 1·4603 67·896 1·4604 67·938 1·4605 67·979 1·4606 68·021 1·4607 68·063 1·4608 68·104 1·4609 68·146 1·4610 68·187 1·4611 68·229 1·4612 68·270 1·4613 68·312 1·4614 68·353 1·4615 68·395 1·4616 68·436 1·4617 68·478 1·4618 68·519 1·4619 68·561 1·4620 68·602 1·4621 68·643 1·4622 68·685 1·4623 68·726 1·4624 68·768 1·4625 68·809 1·4626 68·850 1·4627 68·892 1·4628 68·933 1·4629 68·974 1·4630 69·016 1·4631 69·057 1·4632 69·098 1·4633 69·139 1·4634 69·181 1·4635 69·222 1·4636 69·263 1·4637 69·304 1·4638 69·346 1·4639 69·387 1·4640 69·428 1·4641 69·469 1·4642 69·510 1·4643 69·551 1·4644 69·593 1·4645 69·634 1·4646 69·675 1·4647 69·716 1·4648 69·757 1·4649 69·798 1·4650 69·839 1·4651 69·880 1·4652 69·921 1·4653 69·962 1·4654 70·003 1·4655 70·044 1·4656 70·085 1·4657 70·126 1·4658 70·167 1·4659 70·208 1·4660 70·249 1·4661 70·290 1·4662 70·331 1·4663 70·372 1·4664 70·413 1·4665 70·453 1·4666 70·494 1·4667 70·535 1·4668 70·576 1·4669 70·617 1·4670 70·658 1·4671 70·698 1·4672 70·739 1·4673 70·780 1·4674 70·821 1·4675 70·861 1·4676 70·902 1·4677 70·943 1·4678 70·984 1·4679 71·024 1·4680 71·065 1·4681 71·106 1·4682 71·146 1·4683 71·187 1·4684 71·228 1·4685 71·268 1·4686 71·309 1·4687 71·349 1·4688 71·390 1·4689 71·431 1·4690 71·471 1·4691 71·512 1·4692 71·552 1·4693 71·593 1·4694 71·633 1·4695 71·674 1·4696 71·714 1·4697 71·755 1·4698 71·795 1·4699 71·836 1·4700 71·876 1·4701 71·917 1·4702 71·957 1·4703 71·998 1·4704 72·038 1·4705 72·078 1·4706 72·119 1·4707 72·159 1·4708 72·199 1·4709 72·240 1·4710 72·280 1·4711 72·320 1·4712 72·361 1·4713 72·401 1·4714 72·441 1·4715 72·482 1·4716 72·522 1·4717 72·562 1·4718 72·602 1·4719 72·643 1·4720 72·683 1·4721 72·723 1·4722 72·763 1·4723 72·803 1·4724 72·843 1·4725 72·884 1·4726 72·924 1·4727 72·964 1·4728 73·004 1·4729 73·044 1·4730 73·084 1·4731 73·124 1·4732 73·164 1·4733 73·204 1·4734 73·244 1·4735 73·285 1·4736 73·325 1·4737 73·365 1·4738 73·405 1·4739 73·445 1·4740 73·485 1·4741 73·524 1·4742 73·564 1·4743 73·604 1·4744 73·644 1·4745 73·684 1·4746 73·724 1·4747 73·764 1·4748 73·804 1·4749 73·844 1·4750 73·884 1·4751 73·924 1·4752 73·963 1·4753 74·003 1·4754 74·043 1·4755 74·083 1·4756 74·123 1·4757 74·162 1·4758 74·202 1·4759 74·242 1·4760 74·282 1·4761 74·321 1·4762 74·361 1·4763 74·401 1·4764 74·441 1·4765 74·480 1·4766 74·520 1·4767 74·560 1·4768 74·599 1·4769 74·639 1·4770 74·678 1·4771 74·718 1·4772 74·758 1·4773 74·797 1·4774 74·837 1·4775 74·876 1·4776 74·916 1·4777 74·956 1·4778 74·995 1·4779 75·035 1·4780 75·074 1·4781 75·114 1·4782 75·153 1·4783 75·193 1·4784 75·232 1·4785 75·272 1·4786 75·311 1·4787 75·350 1·4788 75·390 1·4789 75429 1·4790 75469 1·4791 75·508 1·4792 75·547 1·4793 75·587 1·4794 75·626 1·4795 75·666 1·4796 75·705 1·4797 75·744 1·4798 75·784 1·4799 75·823 1·4800 75·862 1·4801 75·901 1·4802 75·941 1·4803 75·980 1·4804 76·019 1·4805 76·058 1·4806 76·098 1·4807 76·137 1·4808 76·176 1·4809 76·215 1·4810 76·254 1·4811 76·294 1·4812 76·333 1·4813 76·372 1·4814 76·411 1·4815 76·450 1·4816 76·489 1·4817 76·528 1·4818 76·567 1·4819 76·607 1·4820 76·646 1·4821 76·685 1·4822 76·724 1·4823 76·763 1·4824 76·802 1·4825 76·841 1·4826 76·880 1·4827 76·919 1·4828 76·958 1·4829 76·997 1·4830 77·036 1·4831 77·075 1·4832 77·113 1·4833 77·152 1·4834 77·191 1·4835 77·230 1·4836 77·269 1·4837 77·308 1·4838 77·347 1·4839 77·386 1·4840 77·425 1·4841 77·463 1·4842 77·502 1·4843 77·541 1·4844 77·580 1·4845 77·619 1·4846 77·657 1·4847 77·696 1·4848 77·735 1·4849 77·774 1·4850 77·812 1·4851 77·851 1·4852 77·890 1·4853 77·928 1·4854 77·967 1·4855 78·006 1·4856 78·045 1·4857 78·083 1·4858 78·122 1·4859 78·160 1·4860 78·199 1·4861 78·238 1·4862 78·276 1·4863 78·315 1·4864 78·353 1·4865 78·392 1·4866 78·431 1·4867 78·469 1·4868 78·508 1·4869 78·546 1·4870 78·585 1·4871 78·623 1·4872 78·662 1·4873 78·700 1·4874 78·739 1·4875 78·777 1·4876 78·816 1·4877 78·854 1·4878 78·892 1·4879 78·931 1·4880 78·969 1·4881 79·008 1·4882 79·046 1·4883 79·084 1·4884 79·123 1·4885 79·161 1·4886 79·199 1·4887 79·238 1·4888 79·276 1·4889 79·314 1·4890 79·353 1·4891 79·391 1·4892 79·429 1·4893 79·468 1·4894 79·506 1·4895 79·544 1·4896 79·582 1·4897 79·620 1·4898 79·659 1·4899 79·697 1·4900 79·735 1·4901 79·773 1·4902 79·811 1·4903 79·850 1·4904 79·888 1·4905 79·926 1·4906 79·964 1·4907 80·002 1·4908 80·040 1·4909 80·078 1·4910 80·116 1·4911 80·154 1·4912 80·192 1·4913 80·231 1·4914 80·269 1·4915 80·307 1·4916 80·345 1·4917 80·383 1·4918 80·421 1·4919 80·459 1·4920 80·497 1·4921 80·534 1·4922 80·572 1·4923 80·610 1·4924 80·648 1·4925 80·686 1·4926 80·724 1·4927 80·762 1·4928 80·800 1·4929 80·838 1·4930 80·876 1·4931 80·913 1·4932 80·951 1·4933 80·989 1·4934 81·027 1·4935 81·065 1·4936 81·103 1·4937 81·140 1·4938 81·178 1·4939 81·216 1·4940 81·254 1·4941 81·291 1·4942 81·329 1·4943 81·367 1·4944 81·405 1·4945 81·442 1·4946 81·480 1·4947 81·518 1·4948 81·555 1·4949 81·593 1·4950 81·631 1·4951 81·668 1·4952 81·706 1·5953 81·744 1·4954 81·781 1·4955 81·819 1·4956 81·856 1·4957 81·894 1·4958 81·932 1·4959 81·969 1·4960 82·007 1·4961 82·044 1·4962 82·082 1·4963 82·119 1·4964 82·157 1·4965 82·194 1·4966 82·232 1·4967 82·269 1·4968 82·307 1·4969 82·344 1·4970 82·381 1·4971 82·419 1·4972 82·456 1·4973 82·494 1·4974 82·531 1·4975 82·569 1·4976 82·606 1·4977 82·643 1·4978 82·681 1·4979 82·718 1·4980 82·755 1·4981 82·793 1·4982 82·830 1·4983 82·867 1·4984 82·905 1·4985 82·942 1·4986 82·979 1·4987 83·016 1·4988 83·054 1·4989 83·091 1·4990 83·128 1·4991 83·165 1·4992 83·202 1·4993 83·240 1·4994 83·277 1·4995 83·314 1·4996 83·351 1·4997 83·388 1·4998 83·425 1·4999 83·463 1·5000 83·500 1·5001 83·537 1·5002 83·574 1·5003 83·611 1·5004 83·648 1·5005 83·685 1·5006 83·722 1·5007 83·759 1·5008 83·796 1·5009 83·833 1·5010 83·870 1·5011 83·907 1·5012 83·944 1·5013 83·981 1·5014 84·018 1·5015 84·055 1·5016 84·092 1·5017 84·129 1·5018 84·166 1·5019 84·203 1·5020 84·240 1·5021 84·277 1·5022 84·314 1·5023 84·351 1·5024 84·388 1·5025 84·424 1·5026 84·461 1·5027 84·498 1·5028 84·535 1·5029 84·572 1·5030 84·609 1·5031 84·645 1·5032 84·682 1·5033 84·719 1·5034 84·756 1·5035 84·792 1·5036 84·829 1·5037 84·866 1·5038 84·903 1·5039 84·939 1·5040 84·976 1·5041 85·013 1·5042 85·049 1·5043 85·086 1·5044 85·123 1·5045 85·159 1·5046 85·196 1·5047 85·233 1·5048 85·269 1·5049 85·306 1·5050 85·343 1·5051 85·379 1·5052 85·416 1·5053 85·452 1·5054 85·489 1·5055 85·525 1·5056 85·562 1·5057 85·598 1·5058 85·635 1·5059 85·672 1·5060 85·708 1·5061 85·744 1·5062 85·781 1·5063 85·817 1·5064 85·854 1·5065 85·890 1·5066 85·927 1·5067 85·963 1·5068 86·000 1·5069 86·036 1·5070 86·072 1·5071 86·109 1·5072 86·145 1·5073 86·182 1·5074 86·218 1·5075 86·254 1·5076 86·291 1·5077 86·327 1·5078 86·363 1·5079 86·399

METHOD 4

MEASUREMENT OF REDUCING SUGARS EXPRESSED AS INVERT SUGARS

(Berlin Institute method)

Scope and field of application

1. The method determines the reducing sugar content expressed as invert sugar in semi-white sugar.

2. Definitions

‘Reducing sugars expressed as invert sugar’: the content of reducing sugars as determined by the method specified.

3. Principle

The sample solution containing reducing sugars is used to reduce a solution of copper II complex. The copper I oxide formed is then oxidized with standard iodine solution, the excess of which is determined by back-titration with standardized sodium thiosulphate solution.

4. Reagents

4.1. Copper II solution (Muller's solution) 4.1.1. Dissolve 35 g of copper II sulphate, pentahydrate (CuSO4.5H2O) in 400 ml of boiling water. Allow to cool. 4.1.2. Dissolve 173 g of sodium potassium tartrate tetrahydrate (Rochelle salt or Seignette salt; KNaC4H4O64H2O) and 68 g of anhydrous sodium carbonate in 500 ml of boiling water. Allow to cool. 4.1.3. Transfer both solutions (4.1.1 and 4.1.2) to a one litre volumetric flask and make up to one litre with water. Add 2 g of activated carbon, shake, allow to stand for several hours and filter through thick filter paper or a membrane filter. If small amounts of copper I oxide appear during storage, the solution should be re-filtered.

4.2. Acetic acid solution 5 mol/litre.

4.3. I odine solution 0·01665 mol/litre (i.e. 0·0333 N, 4·2258 g/litre).

4.4. Sodium thiosulphate solution 0·0333 mol/litre.

4.5. Starch solution: to one litre of boiling water add a mixture of 5 g of soluble starch slurried in 30 ml of water. Boil for three minutes, allow to cool and add, if required, 10 mg of mercury II iodide as a preservative.

5. Apparatus

5.1. Conical flask, 300 ml; precision burettes and pipettes.

5.2. Water-bath, boiling.

6. Procedure

6.1. Weigh a portion of the sample (10 g or less) containing not more than 30 mg of invert sugar in a 300 ml conical flask and dissolve in about 100 ml of water. Pipette 10 ml of the copper II solution (4.1), into the flask containing the sample solution. Mix the contents of the flask by swirling and place it in the boiling water-bath (5.2) for exactly 10 minutes. The level of the solution in the conical flask should be at least 20 mm below the level of the water in the water-bath. Cool the flask rapidly in a stream of cold running water. During this operation the solution should not be stirred otherwise atmospheric oxygen will reoxidize some precipitated copper I oxide. Add 5 ml of 5 mol/litre acetic acid (4.2) by pipette without shaking and immediately add an excess (between 20 and 40 ml) of the iodine solution 0.01665 mol/litre (4.3) from a burette. Stir to dissolve the copper precipitate. Titrate the excess iodine against the sodium thiosulphate solution 0·0333 mol/litre (4.4) using the starch solution (4.5) as indicator. The indicator is added towards the end of the titration.

6.2. Carry out a blank test with water. This is to be carried out with each new copper II solution (4.4). The titration shall not exceed 0·1 ml.

6.3. Carry out a control test under cold conditions with the sugar solution. Allow to stand at room temperature for 10 minutes to permit any reducing agents such as sulphur dioxide which may by present to react.

7. Expression of results.

7.1. Formula and method of calculation

Volume of iodine consumed = ml 0·01665 mol/litre iodine added in excess minus ml 0·0333 mol/litre sodium thiosulphate used in titration.

The volume (in ml) of 0·01665 ml/litre iodine consumed is corrected by subtracting:

7.1.1. The number of ml consumed in the blank test carried out with water (6.2).

7.1.2. The number of ml consumed in the cold test with the sugar solution (6.3).

7.1.3. A value of 2·0 ml for every 10 g of sucrose present in the aliquot used, or a proportionate quantity where the sample contains less than 10 g sucrose (correction for sucrose). After these corrections are made each ml of iodine solution (4.3) which has reacted corresponds to 1 mg of of invert sugar. The invert sugar contents, as a percentage of the sample, is given by the formula: where: V1 = the number of ml of iodine solution (4.3) after correction, mo = the mass, in grams, of the sample used.

7.2. Repeatability

The difference between the results of two determinations when carried out simultaneously or in rapid succession on the same sample, by the same analyst, under the same conditions, shall not exceed 0·02 g per 100 g of sample.

METHOD 5

MEASUREMENT OF REDUCING SUGARS EXPRESSED AS INVERT SUGAR

(Knight and Allen method)

1. Scope and field of application

The method determines the reducing sugar content expressed as invert sugar in:

— sugar or white sugar,

— extra white sugar.

2. Definition

‘Reducing sugars expressed as invert sugar’: the content of reducing sugars as determined by the method specified.

3. Principle

Copper II reagent is added in excess to the sample solution, reduced and the unreduced portion is back-titrated with EDTA solution.

4. Reagents

4.1. Ethylene diamine tetra-acetic acid solution (disodium salt) (EDTA) 0·0025 mol/litre: dissolve 0·930 g of EDTA in water and make up to one litre with water.

4.2. Murexide indicator solution: add 0·25 g of murexide to 50 ml of water and mix with 20 ml of a 0·2 g /100 ml aqueous solution of methylene blue.

4.3. Alkaline copper reagent: dissolve 25 g of anhydrous sodium carbonate and 25 g of potassium sodium tartrate tetrahydrate in about 600 ml of water containing 40 ml of 1.0 mol/litre sodium hydroxide. Dissolve 6·0 g of copper II sulphate pentahydrate (CuSO4.5H2O) in about 100 ml of water, and add to the tartrate solution. Dilute to one litre with water. N.B.: the solution has a limited life (one week).

4.4. Standard invert sugar solution: dissolve 23·750 g of pure sucrose (4.5) in about 120 ml of water in a 250 ml graduated flask, add 9 ml of hydrochloric acid (ζ = 1·16) and allow to stand for eight days at room temperature. Make the solution up to 250 ml and check for completion of hydrolysis by a polarimeter or saccharimeter reading in a 200 mm tube. This should be - 11·80o ± 0·05 oS (see Note 8). Pipette 200 ml of this solution into a 2 000 ml graduated flask. Dilute with water and while shaking (to avoid excessive local alkalinity) add 71·4 ml of sodium hydroxide solution (1 mol/litre) in which 4 g of benzoic acid has been dissolved. Make up to 2 000 ml to give a 1 g/100 ml solution of invert sugar. This solution should be approximately pH 3. This stable stock solution should only be diluted immediately before use.

4.5. Pure sucrose: sample of pure sucrose with an invert sugar content not greater than 0·001 g/100 g.

5. Apparatus

5.1. Test tubes, 150 x 20 mm.

5.2. White porcelain dish.

5.3. Analytical balance, accurate to within 0·1 mg.

6. Procedure

6.1. Dissolve 5 g of sugar sample in 5 ml of cold water in the test tube (5.1). Add 2·0 ml of the copper reagent (4.3) and mix. Immerse the tube in a boiling water bath for five minutes and then cool in cold water.

6.2. Transfer quantitatively the solution in the test tube to the white porcelain dish (5.2) using as little water as possible, add three drops of indicator (4.2) and titrate with EDTA solution (4.1). Vo is the number of ml of EDTA used in the titration. Just before the end-point the colour of the solution changes from green through grey to purple at the end-point. The purple colour will disappear slowly because of oxidation of copper I oxide to copper II oxide at a rate dependent on the concentration of reduced copper present. The end-point of the titration shall therefore be approached fairly rapidly.

6.3. Construct a calibration graph by adding known amounts of invert sugar (as solution 4.4 appropriately diluted) to 5 g of pure sucrose (4.5) and add sufficient cold water so that a total of 5 ml of solution is added. Plot the titration volumes (in ml) against the percentage of invert sugar added to the 5 g of sucrose: the resultant graph is a straight line over the range 0·001 to 0·019 g/100 g invert sugar/100 g sample.

7. Expression of results

7.1. Method of calculation Read on the calibration curve the percentage of invert sugar corresponding to the value Vo ml of EDTA determined when analyzing the sample.

7.2. When a concentration greater than 0·017 g invert sugar/100 g sample is expected in the sample to be analyzed, the sample size taken in Procedure (6.1) must be appropriately reduced but the analysis sample made up to 5 g with pure sucrose (4.5).

7.3. Repeatability The difference between the results of two determinations when carried out simultaneously or in rapid succession on the same sample, by the same, analyst, under the same conditions, shall not exceed 0·005 g per 100 g of sample.

8. Note

Divide by 2·889 to convert oS to polarmetric degrees of arc (precision tubes of 200 mm; light source consisting of a sodium vapour lamp; the instrument must be installed in a room where the temperature may be maintained close to 20 oC).

METHOD 6

DETERMINATION OF REDUCING SUGARS EXPRESSED AS INVERT SUGAR OR DEXTROSE EQUIVALENT

(Luff-Schoorl method)

1. Scope and field of application

The method determines:

1.1. The reducing sugars content expressed as invert sugar in: — sugar solution, — white sugar solution, — invert sugar solution, — white invert sugar solution, — invert sugar syrup, — white invert sugar syrup.

1.2. The reducing sugar content, expressed and calculated (on the dry matter) as the dextrose equivalent in: — glucose syrup, — dried glucose syrup

1.3. The reducing sugar content expressed as D-glucose in: — dextrose monohydrate, — dextrose anhydrous

2. Definition

‘Reducing sugars expressed as invert sugars, D-glucose or dextrose equivalent’: the content of reducing sugars expressed or calculated as invert sugar, D-glucose or dextrose equivalent as determined by the method specified.

3. Principle

The reducing sugars in the sample (clarified if necessary) are heated to boiling point under standardized conditions with a copper II solution, which is partially reduced to copper I. The excess copper II is subsequently determined iodometrically.

4. Reagents

4.1. Carrez solution I: dissolve 21·95 g of zinc acetate dihydrate (Zn(CH3COO)2.2H2O) (or 24 g of zinc acetate trihydrate (Zn(CH3COO)2.3H2O) and 3 ml of glacial acetic acid in water and make up to 100 ml with water.

4.2. Carrez solution II: dissolve 10·6 g of potassium hexacyanoferrate IT trihydrate K4 [Fe(CN)6]. 3H2O in water and make up to 100 ml with water.

4.3. Luff-Schoorl reagent: prepare the following solutions: 4.3.1. Copper II sulphate solution: dissolve 25 g of iron-free copper II sulphate pentahydrate (CuSO4.5H2O) in 100 ml water. 4.3.2. Citric acid solution: dissolve 50 g of citric acid monohydrate (C6H8O7.H2O) in 50 ml of water. 4.3.3. Sodium carbonate solution: dissolve 143.8 g of anhydrous sodium carbonate in about 300 ml of warm water and allow to cool. 4.3.4. Add the citric acid solution (4.3.2) to the sodium carbonate solution (4.3.3) in a one litre volumetric flask with gentle swirling. Swirl until effervescence ceases and then add the copper II sulphate solution (4.3.1) and make up to 1 000 ml with water. Allow the solution to stand overnight and then filter if necessary. Check the molarity of the reagent thus obtained by the method described in 6.1 (Cu 0·1 mol/litre; Na2CO3 1 mol/litre).

4.4. Sodium thiosulphate solution, 0·1 mol/litre.

4.5. Starch solution: to one litre of boiling water add a mixture of 5 g of soluble starch slurried in 30 ml of water. Boil for three minutes, allow to cool and add, if required, 10 mg of mercury II iodide as a preservative.

4.6. Sulphuric acid, 3 mol/litre.

4.7. Potassium iodide solution, 30% (m/v).

4.8. Pumice chips, boiled in hydrochloric acid, washed free of acid with water and then dried.

4.9. Isopentanol

4.10. Sodium hydroxide, 0·1 mol/litre.

4.11. Hydrochlorie acid, 0·1 mol/litre.

4.12. Phenolphthalein solution, 1% (m/v) in ethanol.

5. Apparatus

5.1. Conical flask, 300 ml, fitted with a reflux condenser.

5.2. Stop-watch.

6. Procedure

6.1. Standardization of the Luff-Schoorl reagent (4.3) 6.1.1. To 25 ml of Luff-Schoorl reagent (4.3) add 3 g of potassium iodide and 25 ml of 3 mol/litre sulphuric acid (4.6). Titrate with 0·1 mol/litre sodium thiosulphate (4.4) using starch solution (4.5) as indicator added towards the end of the titration. If the volume of 0·1 mol/litre sodium thiosulphate used is not 25 ml the reagent must be made up afresh. 6.1.2. Pipette 10 ml of the reagent into a 100 ml volumetric flask and dilute to volume with water. Pipette 10 ml of the diluted reagent into 25 ml of 0·1 mol/litre hydrochloric acid (4.11) in a conical flask and heat for one hour in a boiling water-bath. Cool, make up to the original volume with freshly boiled water and titrate with 0·1 mol/litre sodium hydroxide (4.10) using phenolphthalein (4.12) as indicator. The volume of 0·1 mol/litre sodium hydroxide (4.10) used must be between 5·5 and 6·5 ml. 6.1.3. Titrate 10 ml of the diluted reagent (6.1.2) with 0·1 mol/litre hydrochloric acid (4.11) using phenolphthalein (4.12) as indicator. The end-point is characterized by the disappearance of the violet colour. The volume of 0·1 mol/litre hydrochloric acid (4.11) used must be between 6·0 and 7·5 ml. 6.1.4. The pH of the Luff-Schoorl reagent must be between 9·3 and 9·4 at 20 oC.

6.2. Preparation of the solution 6.2.1. Accurately weigh, to the nearest 1 mg, 5 g of the sample and transfer quantitatively to a 250 ml volumetric flask, with 200 ml water. Clarify, if necessary, by adding 5 ml of Carrez solution I (4.1) followed by 5 ml of Carrez solution II (4.2). Mix after each addition. Make up to 250 ml with water. Mix well. Filter if necessary. 6.2.2. Dilute the solution (6.2.1) so that 25 ml of the solution contains not less than 15 mg and not more than 60 mg of reducing sugars expressed as glucose.

6.3. Titration by the Luff-Schoorl method Pipette 25 ml of Luff-Schoorl reagent (4.3) into a 300 ml conical flask (5.1). Pipette 25 ml of the sugar solution (6.2.2) into the conical flask and introduce two pumice chips (4.8). Fit a reflux condenser to the conical flask (5.1) and immediately place the apparatus on an asbestos wire gauze over a Bunsen flame. The gauze shall have a hole cut in the asbestos part of the same diameter as the base of the flask. Heat the liquid to boiling point over a period of about two minutes and simmer gently for exactly 10 minutes. Cool immediately in cold water and after five minutes titrate as follows: Add 10 ml of potassium iodide solution (4.7) then immediately add with caution (because of effervescence) 25 ml of 3 mol/litre sulphuric acid (4.6). Titrate with 01 mol/litre sodium thiosulphate solution (4.4) until the solution is almost colourless, then add a few ml of starch solution (4.5) as indicator and continue titrating until the blue colour disappears. Carry out a control test, using 25 ml of water in place of the 25 ml of sugar solution (6.2.2).

7. Expression of results

7.1. Formula and method of calculation

From the table below, find (interpolating if necessary) the weight of glucose or of invert sugar in mg corresponding to the difference between the two titration readings, expressed in ml of 0·1 mol/litre sodium thiosulphate.

Express the result in terms of invert sugar or D-glucose as percentage (m/m) of the dry matter.

7.2. Repeatability

The difference between the results of two titrations when carried out simultaneously or in rapid succession on the same sample by the same analyst, under the same conditions, shall not exceed 0·2 ml.

8. Note

A small volume of isopentanol (4·9) may be added before acidifying with sulphuric acid to reduce foaming.

Table of values according to Luff-Schoorl reagent

0·1 mol/litre Na2S2O3 Glucose, fructose, invert sugars C6H12O6 ml mg difference 1 2·4 2 4·8 2·4 3 7·2 2·4 4 9·7 2·5 5 12·2 2·5 6 14·7 2·5 7 17·2 2·5 8 19·8 2·6 9 22·4 2·6 10 25·0 2·6 11 27·6 2·6 12 30·3 2·7 13 33·0 2·7 14 35·7 2·7 15 38·5 2·8 16 41·3 2·8 17 44·2 2·9 18 47·1 2·9 19 50·0 2·9 20 53·0 3·0 21 56·0 3·0 22 59·1 3·1 23 62·2 3·1

METHOD 7

MEASUREMENT OF REDUCING SUGARS EXPRESSED AS INVERT SUGAR

(Lane and Eynon constant volume modification)

1. Scope and field of application

The method determines the reducing sugars, expressed as invert sugar, in:

— sugar solution,

— white sugar solution,

— invert sugar solution,

— white invert sugar solution,

— invert sugar syrup,

— white invert sugar syrup.

2. Definition

‘Reducing sugars expressed as invert sugar’: the content of reducing sugars as determined by the method specified.

3. Principle

The sample solution is titrated at the boiling point against a specified volume of Fehling's solution, using methylene blue as internal indicator.

4. Reagents

4.1. Fehling's solution: 4.1.1. Solution A: Dissolve 69·3 g of copper II sulphate pentahydrate (CuSO4.5H2O) in water and make up to 1 000 ml. 4.1.2. Solution B: Dissolve 346·0 g of double sodium potassium tartrate tetrahydrate (KNaC4H4O6.4H2O) with 100·0 g of sodium hydroxide in water and make up to 1 000 ml. The clear solution should be decanted from a sediment that may form from time to time. Note: These two solutions should be stored in brown or amber bottles.

4.2. Sodium hydroxide solution, 1 mol/litre.

4.3. Standard invert sugar solution: dissolve 23·750 g of pure sucrose in about 120 ml of water in a 250 ml graduated flask, add 9 ml of hydrochloric acid (ζ = 1·16) and allow to stand for eight days at room temperature. Make the solution up to 250 ml and check for completion of hydrolysis by a polarimeter or saccharimeter reading in a 200 mm tube. This should be - 11·80o ± 0·05 oS (see note 8). Pipette 200 ml of this solution into a 2 000 ml graduated flask. Dilute with water and while shaking (to avoid excessive local alkalinity) add 71·4 ml of sodium hydroxide solution (1 mol/litre) (4.2) in which 4 g of benzoic acid has been dissolved. Make up to 2 000 ml to give a 1 g/100 ml solution of invert sugar. This solution should be a pH of approximately 3. This stable stock solution should only be diluted immediately before use. To make up the 0·25 g/100 ml invert sugar solution, fill a 250 ml graduated flask to the mark with the stock 1 g/100 ml invert solution at 20 oC. Wash the contents of this flask into a 1 000 ml graduated flask and dilute to the mark with water again at 20 oC.

4.4. Methylene blue solution, 1 g/100 ml.

5. Apparatus

5.1. Narrow-necked laboratory boiling flasks, 500 ml.

5.2. Burette, 50 ml, with tap and offset tip, graduated to 0·05 ml.

5.3. Pipettes graduated at 20, 25 and 50 ml.

5.4. One mark volumetric flasks, 250, 1 000 and 2 000 ml.

5.5. A heating device, suitable for maintaining boiling according to the conditions described in 6.1, permitting the observation of the end-point colour change without the necessity of removing the boiling flask (5.1) from the source of heat.

5.6. Stop-watch, indicating to within at least one second.

6. Procedure

6.1. Standardization of Fehling's solution

6.1.1. Pipette 50 ml of solution B (4.1.2) and then 50 ml of solution A (4.1.1) into a clean dry beaker and mix well.

6.1.2. Rinse and fill the burette with 0·25 % (0·25 g/100 ml) standard invert sugar solution (4.3).

6.1.3. Pipette a 20 ml aliquot of the mixed solutions A and B (6.1.1) into a 500 ml boiling flask (5.1). Add 15 ml of water to the flask. Run in, from the burette, 39 ml of the invert sugar solution, add a small quantity of anti-bumping granules and mix the contents of the flask by gentle swirling.

6.1.4. Heat the flask and contents till boiling and allow to boil for exactly two minutes; the flask must not be removed from the heat source during the course of the rest of the procedure, or allowed to cease boiling. Add three or four drops of methylene blue solution (4.4) at the end of the two-minute boiling period: the solution should be a definite blue colour.

6.1.5. Continue the standardization by adding, from the burette, the standard invert sugar solution in small increments, initially of 0·2 ml; then 0·1 ml and finally in single drops until the end-point is reached. This is indicated by the disappearance of the blue colour imparted by the methylene blue. The solution has then assumed the reddish colour associated with a suspension of copper I oxide.

6.1.6. The end-point should be reached at the end of three minutes from when the solution started to boil. The final titre, Vo, shall be between 39·0 and 41·0 ml. If Vo lies outside these limits, adjust the copper concentration of Fehling's solution A (4.1.1) and repeat the standardization process.

6.2. Preparation of sample solutions

The concentration of the sample test solution should be such that it contains between 250 and 400 mg invert sugar per 100 ml.

6.3. Preliminary test

6.3.1. A preliminary test must be carried out to ensure that the quantity of water to be added to the 20 ml of mixed solutions A and B is sufficient to ensure that a final volume after titration of 75 ml is obtained. The same procedure as described in 6.1.4 is carried out except that the sample solution is used instead of the standard invert sugar solution, i.e. 25 ml of the sample solution is run into the flask from the burette. 15 ml of water is added, and the solution is allowed to boil for two minutes and then titrated until the end-point is reached as described in 6.1.5.

6.3.2. If, after the addition of the methylene blue solution, the reddish colour persists, the sample solution used is too concentrated. In this case, the test is discarded but repeated using a less concentrated sample solution. If more than 50 ml of sample solution are required to obtain the reddish colour, a more concentrated solution of the sample must be used. Calculate the quantity of water to be added by subtracting the volumes of mixed Fehling's solution (20 ml) and of the sample solution from 75 ml.

6.4. Final analysis of sample solution

6.4.1. Pipette into the boiling flask 20 ml of mixed Fehling's solution and the quantity of water determined as in 6.3.

6.4.2. Add, from the burette, the observed titre of the sample solution (as determined in 63) less 1 ml. Add some anti-bumping granules, mix the contents of the flask by swirling, boil the flask and contents and titrate as previously (6.3). The end-point should be reached one minute from the time of addition of the methylene blue solution. Final titre = V1.

7. Expression of results

7.1. Formula and method of calculation

The reducing sugars content of the sample calculation as invert sugar, is given by:

% reducing sugars (as invert sugar =

where:

C = the concentration of the sample test solution in g per 100 ml. Vo = the volume in ml of the standard invert solution used in the standardization titration, V1 = the volume in ml of the sample test solution used in the accurate analysis in 6.4.2, f = the correction factor to take account of the sucrose concentration in the sample test solution. Values are shown in the table below: Sucrose (g in boiling mixture) Correction factor f 0 1·000 0·5 0·982 1·0 0·971 1·5 0·962 2·0 0·954 2·5 0·946 3·0 0·939 3·5 0·932 4·0 0·926 4·5 0·920 5·0 0·915 5·5 0·910 6·0 0·904 6·5 0·898 7·0 0·893 7·5 0·888 8·0 0·883 8·5 0·878 9·0 0·874 9·5 0·869 10·0 0· 64

Corrections for varying sucrose contents of the sample test solution may be calculated from the table by interpolation.

Note:

The approximate sucrose concentration may be found by subtraction of the dissolved solids concentration due to the invert sugar (estimated for the purposes of this calculation f as 1.0), from the total dissolved solids concentration, expressed as sucrose, obtained from the refractive index of the solution using method three of this document.

7.2. Repeatability

The difference between the results of two determinations, carried out simultaneously or in rapid succession on the same sample by the same analyst under the same conditions, shall not exceed 1·0 % of their arithmetic mean.

8. Note

Divide by 2·889 to convert oS to polarmetric degrees of arc (precision tubes of 200 mm; light source consisting of a sodium vapour lamp; the instrument must be installed in a room where the temperature may be maintained close to 20 oC).

METHOD 8

DETERMINATION OF DEXTROSE EQUIVALENT

(Lane and Eynon constant)

1. Scope and field of application

This method determines the dextrose equivalent of:

— glucose syrup,

— dried glucose syrup,

— dextrose monohydrate,

— dextrose anhydrous.

2. Definition

2.1. ‘Reducing power’: the reducing sugar content, determined by the method specified, expressed in terms of anhydrous dextrose (D-glucose) and calculated as a percentage by mass of the sample.

2.2. ‘Dextrose equivalent’: the reducing power, calculated as a percentage by mass of the dry matter in the sample.

3. Principle

The test solution is titrated at the boiling point against a specified volume of mixed Fehling's solution, under strictly specified conditions, using methylene blue as an internal indicator.

4. Reagents

4.1. Fehling's solution:

4.1.1. Solution A: Dissolve 69·3 g of copper II sulphate pentahydrate (CuSO4.5H2O) in water and make up to volume in a 1 000 ml volumetric f ask.

4.1.2. Solution B: Dissolve 346·0 g of sodium potassium tartrate tetrahydrate (KNaC4H4O6.4H2O) and 100 g of sodium hydroxide in water. Make up to volume in a 1 000 ml volumetric flask. Decant the clear solution from any sediment that may from time to time form. Note: These two solutions (4.1.1 and 4.1.2) should be stored in brown or amber bottles.

4.1.3. Preparation of the mixed Fehling's solution Pipette 50 ml of solution B (4.1.2) and then 50 ml of solution A (4.1.1) into a clean dry beaker and mix well. Note: Mixed Fehling's solution shall not be stored but made up afresh every day and standardized (6.1).

4.2. Anhydrous dextrose (D-glucose) (C6H12O6)

This material shall be dried before use for four hours in a vacuum oven at 100 ± 1 oC or less, and an internal pressure of approximately 10 kPa (103 mbar).

4.3. Standard dextrose solution, 0·600 g/100 ml

Weigh, to the nearest 0·1 mg, 0·6 g of anhydrous dextrose (4.2), dissolve it in water, transfer the solution quantitatively into a 100 ml volumetric flask (5.4), dilute to the mark and mix.

This solution shall be freshly prepared on each day of use.

4.4. Methylene blue solution, 0·1 g/100 ml

Dissolve 0·1 g of methylene blue in 100 ml water.

5. Apparatus

5.1. Narrow necked laboratory boiling flasks, 250 ml.

5.2. Burette, 50 ml, with tap and offset tip, graduated to 0·05 ml.

5.3. One mark pipettes, 25 ml and 50 ml.

5.4. One mark volumetric flasks, 100 and 500 ml.

5.5. A heating device, suitable for maintaining boiling according to the conditions described in 6·1, permitting the observation of the end-point colour change without the necessity of removing the boiling flask (5.1) from the source of heat (see 6.1, note 3).

5.6. A stop-watch, indicating to at least the nearest second.

6. Procedure

6.1. Standardization of the Fehling's solution

6.1.1. Pipette 25 ml of Fehling's solution (4.1.3) into a clean, dry boiling flask (5.1).

6.1.2. Fill the burette (5.2) with standard dextrose solution (4.3) and adjust the meniscus to the zero mark.

6.1.3. Run into the boiling flask (5.1) from the burette 18 ml of standard dextrose solution (4.3). Swirl the flask to mix contents.

6.1.4. Place the boiling flask on the heating device (5.5), previously adjusted so that boiling commences in 120 ± 15 seconds. The heating device shall not be further adjusted during the whole of the titration (see note 1).

6.1.5. When boiling commences, start the stop-watch from zero.

6.1.6. Boil the contents of the flask for 120 seconds, as timed by the stop-watch. Add 1 ml of methylene blue solution (4.4) towards the end of this period.

6.1.7. After boiling has continued for 120 seconds (by the stop-watch) start adding standard dextrose solution to the boiling flask (5.1) from the burette (6.1.2) in 0·5 ml increments until the colour of the methylene blue is discharged (see notes 2 and 3). Note the total volume of standard dextrose solution added up to and including the penultimate 0·5 ml increment (X ml).

6.1.8. Repeat 6.1.1 and 6.1.2.

6.1.9. Run into the boiling flask (5.1) from the burette a volume of standard dextrose solution equal to (X-0·3) ml.

6.1.10 Repeat 6.1.4, 6.1.5 and 6.1.6.

6.1.11. After boiling has continued for 120 seconds (by the stop-watch), start adding standard dextrose solution to the boiling flask (5.1) from the burette, initially in 0·2 ml increments and finally dropwise, until the colour of the methylene blue is just discharged. Towards the end of this action the time between successive additions of standard dextrose solution shall be 10 to 15 seconds. These additions shall be completed within 60 seconds, making the total time to boiling no longer than 180 seconds. A third titration with a slightly larger, appropriately adjusted, initial addition of standard dextrose solution (6.1.9) may be necessary to achieve this.

6.1.12. Note the volume (Vo ml) of standard dextrose solution used up to the end-point of the final titration (see note 4).

6.1.13. Vo shall be between 19·0 and 21·0 ml standard dextrose solution (4.3). If Vo lies outside these limits, adjust the concentration of the Fehling's solution A (4.1.1) appropriately and repeat the standardization process.

6.1.14. For the day-to-day standardization of the mixed Fehling's solution, as Vo is known with accuracy, a single titration only is necessary, using an initial addition of (Vo — 0·5) ml standard dextrose solution.

Note 1:

This ensures that once boiling has commenced the evolution of steam is brisk and continuous throughout the whole of the titration process, thus preventing to the maximum possible extent the entrance of air into the titration flask with consquent re-oxidation of its contents.

Note 2:

The disappearance of the colour of the methylene blue is best seen by looking at the upper layers and the meniscus of the contents of the titration flask, as these will be relatively free from the precipiated, red copper I oxide. The colour disappearance is more easily seen when indirect lighting is used. A white screen behind the titration flask is helpful.

Note 3:

The burette should be isolated as much as possible from the source of heat during the determination.

Note 4:

As there is always a personal factor involved, each operator shall carry out his own standardization titration and use his own value of Vo in the calculation (7.1).

6.2. Preliminary examination of the prepared sample

6.2.1. Unless the reducing power (2.1) of the prepared sample is known approximately, it is necessary to carry out a preliminary examination in order to obtain an approximate figure for it so that the mass of the test portion (6.3) can be calculated. This examination is carried out as follows:

6.2.2. Prepare a 2% m/v solution of the sample ‘Z’, having an estimated value.

6.2.3. As 6.1.2, using the sample solution (6.2.2) in place of the standard dextrose solution.

6.2.4. As 6.1.1.

6.2.5. As 6.1.3, using 10·0 ml sample solution instead of 18·0 ml standard dextrose solution.

6.2.6. As 6.1.4.

6.2.7. Heat the contents of the flask to boiling. Add 1 ml methylene blue solution (4.4).

6.2.8. Immediately boiling has started, start the stop-watch (5.6) from zero and commence adding sample solution to the flask from the burette in 1·0 ml increments at intervals of approximately 10 seconds until the blue colour of the methylene blue is discharged. Note the total volume of sample solution added up to and including the penultimate increment (Y ml).

6.2.9. ‘Y’ must not exceed 50 ml. If it does, increase the concentration of the sample solution and repeat the titration.

6.2.10. The approximate reducing power of the prepared sample in percent by mass is given by:

6.3. Test portion

Weigh out, to the nearest 0·1 mg, a mass of the prepared sample (mg) which contains between 2·85 and 3·15 g reducing sugars, expressed as anhydrous dextrose (D-glucose) using in the calculation either known approximate figure for the reducing power (2.1) or the approximate figure obtained in 6.2.10.

6.4. Test solution

Dissolve the test portion in water and make up to 500 ml in a volumetric flask.

6.5. Determination

6.5.1. As 6.1.1.

6.5.2. Fill the burette (5.2) with test solution (6.4) and adjust the meniscus to the zero mark.

6.5.3. Run into the boiling flask from the burette 18·5 ml test solution. Swirl the flask to mix the contents.

6.5.4. As 6.1.4.

6.5.5. As 6.1.5.

6.5.6. As 6.1.6.

6.5.7. As 6.1.7, using test solution in place of standard dextrose solution.

6.5.8. As 6.1.8.

6.5.9. As 6.1.9, using test solution in place of standard dextrose solution.

6.5.10. As 6.1.10.

6.5.11. As 6.1.11, using test solution in place of standard dextrose solution.

6.5.12. Note the volume (V1) of test solution used up to the end-point of the final titration.

6.5.13. V1 shall be between 19·0 and 21·0 ml test solution. If V1 lies outside these limits, adjust the concentration of the test solution appropriately and repeat 6.5.1 to 6.5.12.

6.5.14. Carry out two determinations on the same test solution.

6.6. Dry matter content

Determine the dry matter content of the prepared sample by method 2.

7. Expression of results

7.1. Formulae and method of calculation

7.1.1. Reducing power The reducing power, calculated as a percentage by mass of the prepared sample, is given by: where: V0 = the volume, in ml, of the standard dextrose solution (4.3) used in the standardization titration (6.1), V1 = the volume, in ml, of the test solution (6.4) used in the determination titration (6.5), M = the mass, in grams, of the test portion (6.3) used to make 500 ml test solution.

7.1.2. Dextrose equivalent The dextrose equivalent, calculated as a percentage by mass of the dry matter in the prepared sample, is given by: where: RP = the reducing power, calculated as a percent by mass of the prepared sample (7.1.1), D = the dry matter content of the prepared sample in percent by mass.

7.1.3. Take as the result the arithmetic mean of the two determinations provided that the requirement concerning repeatability (7.2) is satisfied.

7.2. Repeatability

The difference between the results of two determinations when carried out simultaneously or in rapid succession on the same sample, by the same analyst, under the same conditions, shall not exceed 10 % of their arithmetic mean.

METHOD 9

DETERMINATION OF SULPHATED ASH

1. Scope and field of application

The method determines the sulphated ash content in:

— glucose syrup,

— dried glucose syrup,

— dextrose monohydrate,

— dextrose anhydrous.

2. Definition

‘Sulphated ash content’: the content of sulphated ash as determined by the method specified.

3. Principle

The residual mass of a test portion is determined after incineration in an oxidizing atmosphere at 525 oC in the presence of sulphuric acid and calculated as a percentage by mass of the sample.

4. Reagents

4.1. Sulphuric acid, dilute solution: slowly and cautiously add 100 ml of concentrated sulphuric acid (density at 20 oC = 1·84 g/ml; 96 % m/m) to 300 ml water with stirring and cooling.

5. Apparatus

5.1. Electric muffle furnace, equipped with a pyrometer and capable of operating at a temperature of 525 ± 25 oC.

5.2. Analytical balance, accurate to 0·1 mg.

5.3. Ashing crucibles, platinum or quartz, of suitable capacity.

5.4. Desiccator, containing freshly activated silica gel or an equivalent desiccant with a water content indicator.

6. Procedure

Heat a crucible (5.3) to the ashing temperature, cool in a desiccator and weigh. Accurately weigh, to the nearest 01 mg, 5 g of glucose syrup or dried glucose syrup, or about 10 g of dextrose monohydrate or dextrose anhydrous into the crucible.

Add 5 ml of sulphuric acid solution (4.1) (see note 8.1) and carefully heat the sample in the crucible over a flame or on a hotplate until it is completely carbonized. This carbonization process, during which vapours are burnt off from the sample (see note 8.2), should be carried out in a fume cupboard.

Place the crucible (5.3) in the muffle furnace (5.1) heated to 525 ± 25o C until a white ash is obtained. This normally takes two hours (see note 8.3).

Allow the sample to cool for about 30 minutes in a desiccator (5.4) and then weigh.

7. Expression

7.1. Formula and method of calculation

The sulphated ash content expressed as a percentage by mass of the sample to be analyzed is given by:

where:

m1 = the mass, in grams, of the ash, m0 = the mass, in grams, of the test portion.

7.2. Repeatability

The difference between the results of two determinations when carried out simultaneously or in rapid succession on the same sample, by the same analyst, under the same conditions, shall not exceed 2% of their arithmetic mean.

8. Notes

8.1. The sulphuric acid is added in small quantities to prevent excessive foaming.

8.2. Every relevant precaution must be taken during the first carbonization to prevent losses of sample or of ash through excessive swelling of the sample.

8.3. If the sample is difficult to ash completely (i.e. black particles remain) the crucible should be removed from the muffle furnace and the residue moistened, after cooling, with a few drops of water before being returned to the furnace.

METHOD 10

DETERMINATION OF POLARIZATION

1. Scope and field of application

The method determines the polarization in:

— semi-white sugar,

— sugar or white sugar,

— extra-white sugar.

2. Definition

The polarization is the rotation of the polarized light plane by a sugar solution with 26 g of sugar per 100 ml contained in a tube of 200 mm in length.

3. Principle

The polarization is determined by using a saccharimeter or a polarimeter according to the conditions described in the following method.

4. Reagents

4.1. Clarification agent: basic lead acetate solution. Add 560 g of dry basic lead acetate to about 1 000 ml of freshly boiled water. Boil the mixture for 30 minutes and then leave it to stand overnight. Decant the supernatant liquid and dilute with freshly boiled water to obtain a solution of density of 1·25 g/ml, at 20 oC. Protect this solution from contact with air.

4.2. Diethyl ether

5. Apparatus

5.1. Saccharimeter graduated for the normal weight of 26 g of sucrose, or polarimeter This instrument must be installed in a room where the temperature may be maintained close to 20 oC. Calibrate the instrument against standard quartz plates.

5.2. Light source, consisting of a sodium vapour lamp.

5.3. Precision polarimeter tubes, length 200 mm, error not exceeding ± 0·02 mm.

5.4. Analytical balance, accurate to within 0·1 mg,

5.5. Individually calibrated 100 ml volumetric flasks stoppered. Flasks with a real capacity in the range 100·00 ± 0·01 ml may be used without correction. Flasks with a capacity outside those limits must be used with an appropriate correction to adjust the capacity to 100 ml.

5.6. Water-bath, controlled thermostatically at 20 ± 0·1 oC.

6. Procedure

6.1. Preparation of the solution

Weigh as quickly as possible 26 ± 0·002 g of the sample and transfer it quantitatively into a 100 ml volumetric flask (5.5) with approximately 60 ml of water.

Dissolve by swirling but without heating.

Where clarification is necessary, add 0·5 ml of lead acetate reagent (4.1).

Mix the solution by rotating the flask and wash the flask walls, until the volume is such that the meniscus is about 10 mm below the calibration mark.

Place the flask in the water-bath controlled (5.6) at 20 ± 0·1 oC until the temperature of the sugar solution is constant.

Eliminate any bubbles formed at the surface of the liquid with a drop of diethyl ether (4.2).

Make up to volume with water.

Stopper and mix thoroughly by inverting the flask at least three times.

Allow to stand for five minutes.

6.2. Polarization

Maintain the temperature at 20 ± 1 oC for all subsequent operations.

6.2.1. Obtain the zero correction of the apparatus.

6.2.2. Filter the sample through a filter paper. Discard the first 10 ml of the filtrate. Collect the next 50 ml of the filtrate.

6.2.3. Wash the polarimeter tube by rinsing twice with the sample solution to be examined (6.2.2).

6.2.4. Fill the tube carefully at 20 ± 0·1 oC with the sample solution to be examined. Remove all air bubbles when sliding the end-plate into position. Place the filled tube in the cradle of the instrument.

6.2.5. Read the rotation to within 0·05 oS or 0·02 angular degrees. Repeat a further four times. Take the mean of the five readings.

7. Expression of results

7.1. Formula and method of calculation

The results are expressed in degrees S to the nearest 0·1 oS. To convert the angular degrees into degrees S, the following formula is used:

oS = degree of arc × 2·889

7.2. Repeatability

The difference between the results of two determinations when carried out simultaneously or in rapid succession on the same sample, by the same analyst, under the same conditions, and each representing the mean of five readings, must not exceed 0·1 oS.

(1) n values in these tables are calculated from the equation developed by K. Rosenhauer for ICUMSA, programed and computed by Frank G. Carpenter of UDSA, and published in Sugar J. 33, 15-22 (June 1970). Refractive index was measured at 20 oC with 0 line of Na. Brix (% sucrose by weight) was obtained by weighing at 20 oC in air at 760 Torr (mm Hg) pressure and 50% relative humidity. It replaces the previous table, 47.012, 11th edition, taken from Intern. Sugar J. 39, 22s (1937).