المتكون من ( النشا والسكر ) في العلف ومن الكربوهيدرات نستطيع ايجاد الطاقة التمثيلية للعلف .
المواد الكيميائية :ـ
1- 80% ايثانول في دورق حجمي 250مل ( 80مل ايثانول + 20 مل ماء مقطر ) .
2- 52% Perchloric acid
_______52× 100
%70 Perchloric
74.3مل في دورق حجمي 100 مل
-3 محلول 0.1% انترون ( (Anthroneفي 100 مل من ( 76% H 2S04 = 76مل H2S04 + 24 مل مقطر )
4- محلول جلوكوز قياسي ( 100ميكروجرام / مل )
يجفف الجلوكوز في الفرن ثم زن 0.1 جم من الجلوكوز واذابتها في 10مل ماء مقطر ثم يؤخذ منه 25ميكروليتر وتذاب في 250مل ماء مقطر .
الادوات :ـ
1- جهاز Spectrophotometer))
2- جهاز Centrifuge))
3- جهاز ( shaker)
4- ميزان حساس
5- انابيب خاصة لجهاز الطرد المركزي
6- حمام مائي على درجة حرارة C100
7- انابيب باغطية طولها 25سم
8- ورق ترشيح
9- اقماع
طريقة استخلاص السكر
1- وزن 0.2 جم – 0.3 جم من العينة في انبوب جهاز الطرد المركزي
2- اضافة 5 مل من الماء المقطر
3- اضافة 25مل من 80% ايثانول ساخن
4- رجة على الرجاج لمدة 10دقائق
5- وضعة في جهاز الطرد المركزي لمده نصف ساعة
6- اخذ الرائق ووضعة في كاس سعة 100مل
7- اضافة 30مل من الايثانول الساخن على الراسب ثم رجها لمدة 10 دقائق وبعدها توضع في جهاز الطرد المركزي لمده نصف ساعة
8- تأخذ الرائق ونضعه في الكاس السابق (6) ثم اخذ الراسب لتحضير طريقة النشا .
9- وضع الكاس المحتوي على السائل على حرارة هادئة حتى اختفاء رائحة الايثانول ويبقى تقريبا 10% من السائل
10- تكمله الحجم الى 100مل بالماء المقطر باستخدام دورق حجمي سعته 100 مل وترشيح العينة باستخدام قمع وورق الترشيح
# لملاحظة تطور اللون الناتج وذلك من خلال اخذ انابيب الاختبار ووضع فيها
1- 1مل من سائل السكر + 1 مل من ماء مقطر + 10مل انترون
2- 2 مل من سائل السكر + 10مل انترون
3- (بلانك ) 2 مل ماء مقطر + 10مل انترون
غلي الانابيب لمده 12 دقيقة في حمام مائي درجة حرارته 100درجة مئوية تبريدها ثم قراءتها على جهازSpectrophotometer)) على طول موجه 630 وتسمى القراءه 0.D
طريقة النشا :ـ
1- اخذ الراسب الذي نتج من طريقة السكر واضافة عليه
5 مل من الماء المقطر
6.5 مل من Perchloric acid
2- رج العينة لمدة عشر دقائق ثم اضافة 20مل ماء مقطر
3- وضع العينة في جهاز الطرد المركزي لمدة نصف ساعة
4- اخذ السائل الرائق ووضعه في بيكر سعته 100مل
5- اعادة نفس الطريقة على الماده المترسبة
6- ووضع السائل الرائق في نفس البيكر السابق
7- تكملة الحجم الى 100مل بالماء المقطر وذلك باستخدام دورق حجمي سعته 100مل
8- ترشيح باستخدام القمع وورق الترشيح
9- اخذ 1 مل من السائل المرشح ووضعه في دورق حجمي سعته 25مل وتكمله الحجم بالماء المقطر حتى العلامة .
ولملاحظة تطور اللون الناتج وذلك من خلال اخذ
1- 1مل من سائل النشا + 10مل انترون + 1 مل ماء مقطر
2- 2مل من سائل النشا + 10 مل انترون
جهاز قياس الطيف
يقوم جهاز قياس الطيف بتقدير الكربوهيـدرات والدهـون الكلـية الموجودة بالأطعمة
التفاعلات الكيميائية للكربوهيدرات
1) اختبار موليش(( Molisch test
ضع حوالي (3 مل) من المادة الكربوهيدراتية مذابة في ماء مقطر في انبوب اختبار ثم أضف قطرتين من محلول الفا نافثو ل 20% وامزج جيدا ثم على جانب
جدار الأنبوبالمائل اسكب ببطء (1 مل) من حمض الكبريتيك المركز بحيث تنشأ طبقتين سائلتين في الانبوب ,عند وجود مادة كربوهيدراتية تظهر حلقة حمراء تتحول
الى اللون البنفسجي عند نقطة الاتصال بين الطبقتين , رج بلطف وحذر شديد ثم اترك الانبوب ساكناًلمدة دقيقتين ثم خفف بالماء المقطر يظهر راسب بنفسجي في الحال
عند وجود مادة كربوهيدراتية ويرجع اللون الناتج في هذا الاختبار الى تكوين ناتج تكاثف غير ثابت بين مركب الفورفيورال من المادة الكربوهيدراتية والكاشف
الفا نافثول
2) اختبار التفحم(Charing test)
ضع حوالي ( 5و0)جم من المادة الكربوهيدراتية مع (5و0)مل من حمض الكبريتيك المركز في انبوب اختبار وسخن بلطف على لهب صغير يلاحظ حدوث
تفحم مع تصاعد غازات عبارة عن خليط من (ٍSO2,CO,CO2) ويرجع التفحم الى قدرة حمض الكبريتيك على نزع الماء من المادة الكربوهيدراتية
تفاعلات خاصة بالسكريات المختزلة
إن المواد الكربوهيدراتية التي تحتوي على مجموعة كربونيل نشطة سواء في صورة الدهيد او كيتون لها القدرة على اختزال محاليل بعض الايونات المعدنية ومنهـــــا :
• اختزال محلول فهلنج (Fehling)عند اضافة محلول فهلنج 1و2 الى محلول يغلي لمادة سكرية مختزلة يلاحظ اختزال محلول فهلنج بسرعة وإختفاء اللون الأزرق وظهور راسب احمر من أكسيد النحاسوز
• اختزال محلول بندكت (Benedict)
ضع في انبوب اختبار 2 مل محلول بندكت ثم اضف (5و0) مل من محلول السكر المختزل وضع الانبوب في حمام مائي لمدة دقيقتين نلاحظ ظهور
راسب اخضر بالانبوب يتحول للاصفر ثم الاحمر وذلك تبعا لكمية السكر المختزل• اختزال محلول بارفويد(Barfoied)
اضف في انبوب اختبار (3 مل )من محلول بارفويد الى (2مل )من محلول مخفف للسكر المختزل وسخن لمدة دقيقتين في حمام مائي يغلي –
يلاحظ حدوث اختزال لخلات النحاس (الداخلة في تركيب البارفويد ) ويظهر راسب احمر من اكسيد النحاسوز
• اختزال نترات الفضة النوشادرية
ضع (2 مل )من نترات الفضة النوشادرية ثم 1 مل من محلول السكر المختزل يلاحظ اختزال نترات الفضة ببطء ويصبح المحلول ذا لون بني او اسود وعند تسخين
الخليط في حمام مائي يتم الاختزال بسرعة وتتكون مرآة فضية على جدران الانبوب
• اختزال الاوزازون(Osazone)ضع في انبوب اختبار(2 مل )من(2و0 جم محلول الفينيل هيدرازين هيدوكلوريد مع 3و0 جم خلات صوديوم) ثم اضف (1و0 جم) من الجلوكوز
وسخن لمدة (20-30)دقيقة في حمام مائي واترك الأنبوب يبرد ببطء تلاحظ ظهور بلورات صفراء يمكن رؤيتها تحت المجهر لاحظ أن كلا من الفركتوز
والجلوكوز والما نوز تعطي نفس البلورات ذات الشكل الهندسي المعين وذلك لان التفاعل يتم بين ذرة الكربون (1-2) في جزيء السكر وباقي ذرات الكربون
في السكريات الثلاثة متشابهة
يلاحظ أن سكر اللاكتوز وسكر المالتوز يعطيان مشتقات (Osazones) ولكنها تذوب في الماء الساخن ولذلك لا تظهر إلا عند التبريد في حين
أن مشتقات Osazones)) مع السكريات الأخرى لا تذوب في الماء الساخن ولذا تنفصل عندما يكون المحلول ساخنا
اختبارات نوعية لبعض السكريات
1. الجلوكوز :تأثير هيدروكسيد القلويات :عند تسخين الجلوكوز مع محلول (3%) من هيدروكسيد الصوديوم يلاحظ ظهور لون اصفر في باديء الأمر
ويتحول إلى البني ثم الأحمر
2. الفركتوز
أ- اختبار الفورفيورال
إن الدهيد الفورفيورال هو أحد نواتج هدم الفركتوز بالأحماض , ويمكن الكشف عنه بتمييز اللون الأحمر الساطع الذي يعطيه مع خلات الانيلين ولإجراء الاختبار أذب
(3و0) جم من الفركتوز في (5) مل من محلول هيدروكلوريك مخفف بنسبة (3:1) واغلي لمدة دقيقة حيث يتحول المحلول إلى اللون البني الغامق والأبخرة
المتصاعدة من الدهيد الفورفيورال تحول شريط من ورق الترشيح المبلل بخلات الانيلين إلى اللون الأحمر الساطع
ب_ اختبار بينوف (Pinoff test)أذب (1و0) جم من الفركتوز في (10 مل) من الماء المقطر ثم أضف 10 مل من موليبيدات
الامونيوم تركيز 4% ثم أضف قطرات من حمض الخليك الثلجي وسخن في حمام مائي لمدة 5 دقائق تلاحظ ظهور لون أزرق غامق
( الاختبار موجب في غياب الأحماض المعدنية )
ج – اختبار سلوانوف
أضف (5 قطرات ) من سكر الفركتوز إلى 4 مل من كاشف السلوانوف واغلي لمدة 20 ثانية فقط ثم برد يظهر لون احمر في الأنبوب
3. الجالاكتوز
اختبار بينوف
أضف (1و0) جم من الجالاكتوز إلى 10 مل ماء مقطر واجعل السكر يذوب ثم أضف 10 مل من محلول (4%) موليبيدات امونيوم ثم أضف 4 نقط من
حمض الخليك الثلجي وسخن في حمام مائي لمدة (10-)20 دقيقة يظهر لون ازرق باهت
يعطي الجالاكتوز لون ازرق باهت بعد التسخين لمدة 10 دقائق وفي حالة الفركتوز يظهر اللون ازرق غامق بعد الغليان لمدة 5 دقائق فقط
اختبار حمض (mucic)))
في بوتقة من الخزف ضع حوالي ممل من محلول الجالاكتوز ثم اصف 5 نقط من حمض النيتريك المركز – بخر ببطء على حمام مائي حتى يقل حجم المحلول إلى
حوالي 2 مل اترك البوتقة تبرد وبحيث لا تحرك من مكانها وافحص عن وجود بلورات بيضاء تظهر كراسب من حمض وهذا الراسب لا يذوب في الماء ولكن يذوب
في القلويات ويعاد ترسيبة بإضافة حمض إلى المحلول .
اذا فالكربوهيدرات هي مركبات كيميائية عضوية تتكون من الكربون , والهيدروجين , والاكسجين . وتعتبر هذه المركبات من مصادر الطاقة في جسم الكائن الحي,
والمادة التركيبية لعضيات الخلية .
Laboratory Analytical Procedure #002
1. Introduction
1.1 The carbohydrates making up a major portion of biomass samples are polysaccharides composed
primarily of glucose, xylose, arabinose, galactose, and mannose subunits. The polysaccharides
present in a biomass sample can be hydrolyzed to their component sugar monomers by sulfuric
acid in a two-stage hydrolysis process. The sample can then be quantified by ion-moderated
partition HPLC.
1.2 This procedure has been adopted by ASTM as the Standard Test Method for Determination of
Carbohydrates in Biomass by High Performance Liquid Chromatography, E1758-95.
2. Scope
2.1 This method covers the determination of carbohydrates, expressed as the percent of each sugar
present in a hydrolyzed biomass sample. The sample is taken through a primary 72% sulfuric
acid hydrolysis, followed by a secondary dilute-acid hydrolysis.
2.2 Sample material suitable for this procedure include hard and soft woods, herbaceous materials
(such as switchgrass and sericea), agricultural residues (such as corn stover, wheat straw, and
bagasse), waste-paper (such as office waste, boxboard, and newsprint), washed acid- and
alkaline-pretreated biomass, and the solid fraction of fermentation residues. All results are
reported relative to the 105°C oven-dried weight of the sample. In the case of extracted
materials, the results may also be reported on an extractives-free basis.
2.3 All analyses shall be performed according to the guidelines established in the Ethanol Project
Quality Assurance Plan (QAP).
3. References
3.1 Moore, W.E., and D.B. Johnson. 1967. Procedures for the Chemical Analysis of Wood and
Wood Products. Madison, WI: U.S. Forest Products Laboratory, U.S. Department of
Agriculture.
3.2 Ethanol Project Laboratory Analytical Procedure #001, "Standard Method for the Determination
of Total Solids in Biomass".
3.3 Ethanol Project Laboratory Analytical Procedure #003, "Determination of Acid-Insoluble Lignin
in Biomass".
3.4 NREL Ethanol Project Laboratory Analytical Procedure #004, "Determination of Acid-Soluble
Lignin in Biomass
Procedure #002
Issue Date: 8/12/96
Supersedes: 1/30/95
Page 3 of 11
3.5 NREL Ethanol Project Laboratory Analytical Procedure #010, "Standard Method for the
Determination of Extractives in Biomass".
3.6 TAPPI Test Method T264 om-88, "Preparation of Wood For Chemical Analysis." In Tappi Test
Methods. Atlanta, GA: Technical Association of the Pulp and Paper Industry.
3.7 Vinzant, T.B., L. Ponfick, N.J. Nagle, C.I. Ehrman, J.B. Reynolds, and M.E. Himmel. 1994.
"SSF Comparison of Selected Woods From Southern Sawmills." Appl. Biochem. Biotechnol.,
45/46:611-626.
4. Terminology
4.1 Prepared Biomass - Biomass that has been prepared by lyophilization, oven drying, air drying,
and in some instances by extraction, to reduce the moisture content of the sample so it is
suitable for carbohydrate analysis.
4.2 Oven-Dried Weight - The moisture-free weight of a biomass sample as determined by LAP-001,
"Standard Method for Determination of Total Solids in Biomass".
5. Significance and Use
5.1 The percent sugar content is used in conjunction with other assays to determine the total
composition of biomass samples.
6. Interferences
6.1 Samples with high protein content may result in percent sugar values biased low, as a
consequence of protein binding with some of the monosaccharides.
6.2 Test specimens not suitable for analysis by this procedure include acid- and alkaline-pretreated
biomass samples that have not been washed. Unwashed pretreated biomass samples containing
free acid or alkali may change visibly on heating.
7. Apparatus
7.1 Hewlett Packard Model 1090 HPLC, or equivalent, with refractive index detector.
7.2 HPLC columns, BioRad Aminex7 HPX-87C and/or Aminex7 HPX-87P (or equivalent).
7.3 Guard columns, cartridges appropriate for the column used.
Note: Deashing guard column cartridges from BioRad, of the ionic form H+/CO3
%,
are an option when using an HPX-87P column. These cartridges have been found
to be effective in eliminating baseline ramping.
7.4 Analytical balance readable to 0.1 mg.
7.5 Convection ovens with temperature control to 45 ± 3°C and 105 ± 3°C.
7.6 Autoclave capable of maintaining 121 ± 3°C.
7.7 Water bath set at 30 ± 3°C.
7.8 Desiccator containing anhydrous calcium sulfate.
8. Reagents and Materials
8.1 Reagents
8.1.1 High purity sugars for standards (98%+) - two sets of glucose, xylose, galactose,
arabinose,and mannose from different lots or manufacturers.
8.1.2 72% w/w H2SO4 (12.00 ± 0.02 M or specific gravity 1.6389 at 15.6 °C /15.6°C).
8.1.3 Calcium carbonate, ACS reagent grade.
8.1.4 Water, 18 megohm deionized.
8.2 Materials
8.2.1 Glass test tubes, 16x100 mm.
8.2.2 125 mL glass serum bottles, crimp top style, with rubber stoppers and aluminum seals
to fit.
8.2.3 pH paper, suitable to cover the pH range of 4 to 7.
8.2.4 Disposable nylon syringe filters, 0.2 mm.
8.2.5 Disposable syringes, 3 mL.
8.2.6 Autosampler vials, with crimp top seals to fit.
8.2.7 Erlenmeyer flasks, 50 mL.
9. ES&H Considerations and Hazards
9.1 Follow all applicable NREL Laboratory Specific Hygiene Plan guidelines.
9.2 72% H2SO4 is very corrosive and must be handled carefully.
9.3 Use caution when handling hot glass bottles after the autoclave step, as they may have become
pressurized.
10. Sampling, Test Specimens and Test Units
10.1 Test specimens suitable for analysis by this procedure are as follows:
- biomass feedstocks, dried and reduced in particle size, if necessary.
- pretreated biomass, washed free of any residual acid or alkali.
- the solids fraction of fermentation residues.
10.2 The sample must not contain particles larger than 1 mm in diameter. If milling is required to
reduce the particle size of the test specimen, a laboratory mill equipped with a 40 mesh (or
smaller) screen should be used.
10.3 The total solids content of the "as received" test specimen (prior to any drying or extraction
steps) must be determined by LAP-001 in parallel with the carbohydrate analysis. Record this
value as %Tas received.
10.4 Material with a total solids content less than 85%, on a 105°C dry weight basis, will require
drying by lyophilization, oven drying, or air drying prior to milling or analysis. The amount of
moisture lost as a result of the preparation procedure must be determined. This moisture content
is used to calculate the total solids content of the sample based on its preparation and is recorded
as %Tprep. This value is used to correct the weight of the prepped material used in the
carbohydrate analysis, as described in the calculations section. The prepared sample should be
stored in a manner to ensure its moisture content does not change prior to analysis.
Note: Preparing samples for analysis by oven drying can produce hard chunks of
material. This material must then be milled to reduce the size of the large pieces to
less then 1 mm in diameter. The sample is then redried prior to testing.
10.5 Some samples may require extraction prior to analysis, to remove components that may interfere
with the analysis. LAP-010, "Standard Method for the Determination of Extractives in
Biomass", is used to prepare an extractives-free sample with a moisture content suitable for
carbohydrate analysis. As part of this procedure, the percent extractives in the prepared sample,
on a 105°C dry weight basis, is determined. This value, recorded as % extractives, can be used
to convert the % sugar reported on a extractives-free basis to an as received (whole sample)
basis.
10.6 The test specimen shall consist of approximately 0.3 g of sample. The test specimen shall be
obtained in such a manner to ensure that it is representative of the entire lot of material being
tested.
11. Procedure
11.1 This procedure is suitable for air-dried, lyophilized, and extracted biomass samples, as well as
for samples that have been oven dried at a temperature of 45°C or less. It is not suitable for
samples that have been dried at a temperature exceeding 45°
Note: The total solids content of the original sample, %Tas received, must be determined
using LAP-001, prior to any preparatory steps. The total solids content of the sample
based on its preparation, %Tprep , must also be known.
11.2 Determine the total solids content of the prepared or extractives-free biomass sample by LAP-
001 and record this value as %Tfinal .
Note: Samples for total solids determination (LAP-001) must be weighed out at the
same time as the samples for the carbohydrate determination. If this is done later, it
can introduce an error in the calculation because ground biomass can rapidly gain or
lose moisture when exposed to the atmosphere.
11.3 Weigh 0.3 ± 0.01 g of the prepared or extractives-free sample to the nearest 0.1 mg and place
in a 16x100 mm test tube. Record as W1, the initial sample weight in grams. Each sample must
be run in duplicate, at minimum.
11.4 Add 3.00 ± 0.01 mL (4.92 ± 0.01 g) of 72% H2SO4 and use a glass stirring rod to mix for 1
minute, or until the sample is thoroughly wetted.
11.5 Place the test tube in the water bath set at 30 ± 1°C and hydrolyze for 2 hours.
11.6 Stir the sample every 15 minutes to assure complete mixing and wetting.
11.7 Weigh out 0.3 ± 0.01 g of each high purity sugar (predried at 45°C) to the nearest 0.1 mg, and
place each in its own 16x100 mm glass test tube. Add acid, hydrolyze, and stir these sugars as
described in the previous three steps. These sugar recovery standards (SRS) will be taken
through the remaining steps in the procedure in parallel with the samples. The calculated
recovery of the SRSs will be used to correct for losses due to the destruction of sugars during
the hydrolysis process. It may be useful to run selected SRSs in duplicate, particularly if
specific sugars are deemed critical.
11.8 Prepare a method verification standard (MVS) by weighing out 0.3 ± 0.01 g of a well
characterized standard material suitable for analysis. Add acid, hydrolyze, and stir the MVS as
was done with the samples and SRSs (see 11.4-11.6 above). This MVS will be taken through
the remaining steps in the procedure in parallel with the samples and the SRSs, and is used to
test the reproducibility of the method as a whole.
Note: A suitable method verification standard, Populus deltoides, may be obtained
from NIST (research material #8492).
11.9 Upon completion of the two hour hydrolysis step, transfer each hydrolyzate to its own serum
bottle and dilute to a 4% acid concentration by adding 84.00 ± 0.04 mL deionized water. Be
careful to transfer all residual solids along with the hydrolysis liquor. The total weight added
to the tared bottle is 89.22 g (0.3 g sample, 4.92 g 72% H2SO4, and 84.00 g deionized water).
Since the specific gravity of the 4% acid solution is 1.0250 g/mL, the total volume of solution,
VF , is 87.0 mL.
11.10 Stopper each of the bottles and crimp aluminum seals into place.
11.11 Set the autoclave to a liquid cycle to prevent loss of sample from the bottle in the event of a
loose crimp seal. Autoclave the samples in their sealed bottles for 1 hour at 121 ± 3°C.
11.12 After completion of the autoclave cycle, allow the samples to cool for about 20 minutes at room
temperature before removing the seals and stoppers.
11.13 These autoclaved solutions may also be used for the determination of acid-insoluble residue
and/or acid-soluble lignin, in parallel with this carbohydrate determination.
Note: If acid-insoluble lignin and/or acid-soluble lignin determinations are to be
conducted on a sample, the residual solids must be collected by filtering the
hydrolyzate through an ashed and weighed filtering crucible prior to proceeding with
the carbohydrate determination. Refer to LAP-003, "Determination of Acid-
Insoluble Lignin in Biomass", for details. If an acid-soluble lignin determination is
to be conducted, a portion of the filtrate must be reserved for analysis. Acid-soluble
lignin should be analyzed within 24 hours, preferably within 6 hours of hydrolysis.
Refer to the procedure "Determination of Acid-Soluble Lignin in Biomass" (LAP-
004) for details.
11.14 Transfer 20 mL aliquots of each hydrolyzate, or filtrate, to 50 mL Erlenmeyer flasks.
11.15 Neutralize with calcium carbonate to a pH between 5 and 6. Do not over-neutralize. Add the
calcium carbonate slowly with frequent swirling to avoid problems with foaming. Monitor the
pH of the solution with pH paper to avoid over-neutralization.
11.16 Filter the neutralized hydrolyzate using a 3 mL syringe with a 0.2 mm filter attached. One
portion of the hydrolyzate should be filtered directly into a sealable test tube for storage. A
second portion should be filtered directly into an autosampler vial if the hydrolyzate is to be
analyzed without dilution. If the concentration of any of the analytes is expected to exceed the
validated linear range of the analysis, dilute the hydrolyzate as required and filter into an
autosampler vial for analysis.
Note: It is advisable to determine the initial glucose concentration of the sample
using an alternative technique, such as a YSI glucose analyzer, in order to predict
whether or not the glucose in the sample will fall within the linear range of the
analysis.
11.17 The portion of the neutralized hydrolyzate filtered into the test tube should be securely sealed,
labeled, placed in the refrigerator, and reserved in case a repeat analysis is required. The sample
should be stored for no longer than two weeks.
11.18 Prepare a series of sugar calibration standards in deionized water at concentrations appropriate
for creating a calibration curve for each sugar of interest. A suggested scheme for the HPX-87C
column is to prepare a set of multi-component standards containing glucose, xylose, and
arabinose in the range of 0.2 -12.0 mg/mL. For the HPX-87P column, galactose, and mannose
should be included as additional components in the standards. Extending the range of the
calibration curves beyond 12.0 mg/mL will require validation.
11.19 Prepare an independent calibration verification standard (CVS) for each set of calibration
standards, using sugars obtained from a source other than that used in preparing the calibration
standards. The CVS must contain precisely known amounts of each sugar contained in the
calibration standards, at a concentration that falls in the middle of the validated range of the
calibration curve. The CVS is to be analyzed after each calibration curve and at regular
intervals in the HPLC sequence, bracketing groups of samples. The CVS is used to verify the
quality of the calibration curve(s) throughout the HPLC run.
11.20 Analyze the calibration standards, the CVS, the samples, the SRSs, and the MVS by HPLC
using a Biorad Aminex7 HPX-87C or HPX-87P column for glucose, xylose, and arabinose. If
mannose and galactose are also to be determined, a Biorad Aminex7 HPX-87P column must
be used instead. For many analyses, it is useful to run the same samples on both columns and
compare the results. The following instrumental conditions are used for both the HPX-87C and
the HPX-87P columns:
Sample volume: 50 mL.
Eluant: 0.2 mm filtered and degassed, deionized water.
Flow rate: 0.6 mL/min.
Column temperature: 85°C.
Detector: refractive index.
Run time: 20 minutes data collection plus a 15 minute post-run.
12. Calculations
12.1 Create a calibration curve by linear regression analysis for each sugar to be quantified. From
these curves, determine the concentration in mg/mL of the sugars present in each solution
analyzed by HPLC.
12.2 Calculate the amount of sugar recovered from each SRS taken through the two-stage hydrolysis.
This will give an estimate of the amount of each individual sugar destroyed during the
hydrolysis procedure.
Where: %R srs = % recovery of sugar recovery standard
C1 = known concentration of sugar recovery standard before hydrolysis, in
mg/mL
C2 = concentration of sugar recovery standard detected by HPLC after
hydrolysis, in mg/mL
12.3 Use the percent recovery of the appropriate sugar recovery standard to correct sugar
concentration values (in mg/mL) obtained from HPLC for each sugar detected in the hydrolyzed
sample.
Where:
Ccorr = concentration of sugar in hydrolyzed sample corrected for hydrolysis,
in mg/mL
Cspl = concentration of sugar detected in the hydrolyzed sample by HPLC,
in mg/mL
%Rsrs = % recovery of sugar recovery of standard, as determined in the
previous step
12.4 For lyophilized, air dried, or oven dried samples, or for samples requiring no preparation,
calculate the percentage of each sugar present in the sample on an as received 105°C dry weight
basis as follows:
Where: W1 = initial weight of sample, in grams
VF = volume of filtrate, 87.0 mL
Ccorr = concentration of sugar in hydrolyzed sample corrected for loss on hydrolysis,
is determined in previous step, in mg/mL
%Tas received = % total solids content of the original sample (prior to any preparation
steps) on a 105°C dry weight basis, as determined by the LAP-001
%Tprep = % total solids content of the sample as determined during the preparation
of the sample for analysis (by lyophilization, oven-drying, or air drying)
%Tfinal = % total solids content of the prepared sample used in this carbohydrate
analysis, on a 105°C dry weight basis, as determined by the LAP-001
Note: If the sample used in the analysis required no preparation (analyzed as
received), then %Tprep = 100% and %Tfinal = %Tas received.
100%
%R
C =C srs
corr spl ¸
x 100%
100%
%T
W x
xV
1000 mg
1 g
C x
x 100%=
%T
%T
W x
xV
1000 mg
1 g
C x
% Sugar =
final
1
corr F
prep
as received
1
corr F
Procedure #002
Issue Date: 8/12/96
Supersedes: 1/30/95
Page 10 of 11
12.5
12.5 If the biomass was prepared according to the "Standard Method for the Determination of
Ethanol Extractives in Biomass" (LAP-010), first calculate the percentage of each sugar present
on an extractives-free 105EC dry weight basis and then correct this value to an as received
(whole sample) 105°C dry weight basis.
12.5.1
Calculate the percentage of each sugar on an extractives-free basis as follows:
Where: Ccorr = concentration of sugar in hydrolyzed sample corrected for loss on
hydrolysis, as determined above, in mg/mL
VF = volume of filtrate, 87.0 mL
W1 = initial weight of extracted sample, in grams
%Tfinal = % total solids content of the prepared sample used in this
carbohydrate analysis (in this case, extracted sample), on a 105°C dry
weight basis, as determined by the LAP-001
12.5.2 Correct the % sugar value on an extractives-free basis, calculated above, to an as
received (whole sample) 105°C dry weight basis as follows:
Where:
% Sugarextractives-free = % sugar on an extractives-free 105°C dry weight basis,
as determined in the previous step
% extractives = % extractives in the extracted sample as described in the
Standard Method for the Determination of Extractives in Biomass
13. Report
13.1 Report the percent sugar present in the sample, to two decimal places, on a whole sample 105°C
dry weight basis or on an extractives-free basis. Cite the basis used in the report.
13.2 For replicate analyses of the same sample, report the average, standard deviation, and relative
percent difference (RPD).
رد مع اقتباس
رد: تحليل اغذية (تقدير الكربوهيدرات ) 
مواقع النشر (المفضلة)