BIOCHEMISTRY LAB REPORT

AYU LAKSMI PUSPITASARI

BIOLOGY IUP
24020118190151

TOPIC VI
CARBOHYDRATE QUANTITATIVE ANALYSIS

I. Basic Competencies
Practitioners are able to analyze carbohydrate quantitatively on samples.

II. Literature Review

2.1. Carbohydrate Quantitative Analysis
Carbohydrate analysis is important from several perpectives. Qualitative and
quantitative analysis is used to determine compositions of foods, beverages, and their
ingredients. Quantitative analysis ensures that ingredient label present accurate
compositional information. Quantitative analysis ensures that added components are
listed in the proper order on ingredient labels. Quantitative analysis also ensures that
amounts of specific components of consumer interest, for example, β-glucan, are
proper and that caloric contect can be calculated. Both qualitative and quantitative
analysis can be used to authenticate (i.e., to detect adulteration of) food ingredients
and products (Nielsen, 2010).
2.2. Glucose
Glucose is a monosaccharide containing six carbon atoms and an aldehyde group
and is therefore referred to as an aldohexose. The glucose molecule can exist in an
open-chain (acyclic) and ring (cyclic) form, the latter being the result of an
intramolecular reaction between the aldehyde C atom and the C-5 hydroxyl group to
form an intramolecular hemiacetal. In water solution both forms are in equilibrium
and at pH 7 the cyclic one is the predominant. Glucose is a primary source of energy
for living organisms. It is naturally occurring and is found in fruits and other parts of
plants in its free state. In animals glucose arises from the breakdown of glycogen in a
 process known as glycogenolysis. Glucose is synthesized in the liver and kidneys
from non-carbohydrate intermediates, such as pyruvate and glycerol, by a process
known as gluconeogenesis. D-Glucose is found to be associated with 3-methyl-
crotonyl-glycinuria, growth hormone deficiency, and primary hypomagnesemia,
which are inborn errors of metabolism (Human Metabolome Database, 2019).
Glucose, also called dextrose, belongs to a group of carbohydrates known ad
simple sugars (monosaccharides). Glucose has the molecular formula C5H12)6. Is is
found in fruits and honey and is the major free sugar circulating in the blood of higher
animals. It is the source of energy in cell function, and the regulation of its
metabolism is of great importantce (gluconeogenesis) Molecules of starch, the major
energy reserve carbohydraye of plants, consists of thousands of glucose units, as do
those of cellulose. Also composed of glucose is glycogen, the reserve carbohydrte in
most vertebrate and invertebrate animal cells as well as those of numerous fungi and
protozoans (Shendurse and Khedkar, 2016).
2.3. Spectrophotometry
Spectrophotometry is the quantitative measurement of the interaction of
ultraviolet (UV), visible, and ifrared radiation with a material and has an impact on a
wide field of science and technology. The nature of this interaction depends upon the
physical properties of the material, for example, transparent or opaque, smooth or
rough, pure or contaminated, and thin or thick. Thus spectrophotometric measurement
can be used to quatify, in turn, these imporatant physical properties of the material.
The choices of pectrophotometric measurements include spectral reflectance,
transmittance, absorptance, emttance, scattering, and fluorescene and can be classifies
as phenomenological optical properties of the material. Spectrophotometric
measurements can also be used to probe the intrinsic or internal physical nature of the
material, such as its reflective index and extinction coefficient. The pharmaceutical
and chemical undustries use optical absorption and fluorescene measurements to
quantify concentration which is required for accurate dosing and elimination of
contaminants. The interaction of the light with the material gives us an overall
impression of its quality. The spectral, spatial and directional properties permit us to
 identify material, characterize topography, and observe defects without ever coming
into contact with the object (Germer et al, 2014).

III. Methods
3.1. Tools
1. Test tube
2. Test tube rack
3. Erlenmayer
4. Drop pipette
5. Spectrophotometer
6. Laboratory manual book
7. Laboratory temporary report
8. Stationary
3.2. Materials
1. Glucose sample 0.005, 0.01, 0.02, 0.03, 0.04, 0.05 gram
2. DNS reagent
3. Aquades

3.3. Procedure
1. Glucose solution is made by mixing glucose sample and aquades.
2. Glucose sample absorbation then measured using spectrophotometer.
3. Measurement result is made into table and standarized curve based on equation
Y=aX+b
4. All result is documented on temporary report.
IV. Result
4.1.Standardized Curve Table
4.1.1. Sample 1
No Concentration Transmittance Absorbation
1 0,005 7;7;7 1,15
2 0,01 6;6 1,22
4 0,02 7,5 ; 7,5 ; 7,5 1,12
5 0,03 6,5 ; 6 ; 6,5 1,20
(6,33)
6 0,04 4,5 ; 5 ; 6 ; 6 ; 6,5 (5,4) 1,27
7 0,05 1 ; 2 ; 1,5 -
(1,5)

4.1.2. Sample 2
No Concentration Transmittance Absorbation
1 0,005 3,5 ; 3,5 ; 3,5 1,46
2 0,01 3;3;3 1,52
4 0,02 3 ; 3; 2 ; 3 1,56
(2,75)
5 0,03 2,5 ; 3 ; 3 1,55
(2,8)
6 0,04 1;1;1 2
7 0,05 0 ; 0,5 ; 0 2,77
(0,17)
4.2.Standarized Curve

Y = aX + b
Y = 0,4543X + 0,0062
X = concentration of reducing sugar
Y = absorbance axis
V. Discussion
Biochemistry laboratory practice topic VI titled “Carbohydrate Qualitative
Anaylsis” on Thursday, May 23th, 2019 in Biochemistry Laboratory of Science and
Mathematics Faculty, Diponegoro University, was held on the purpose to analyze
carbohydrate quantitatively on samples. The tools used were test tube, test tube rack,
erlenmayer, drop pipette, spectrophotometer, laboratory manual book, laboratory
temporary report, and stationary. The materials used were glucose sample 0.005, 0.01,
0.02, 0.03, 0.04, 0.05 gram, DNS reagent, and aquades.
In this practicum, glucose is available in solid form stored in aluminum foil and
has been weighed according to the weight required. Then, we should dilute the glucose in
tube reaction with aquades based on the comparison requested. Why must dilute?
Because, when enter the spectrophotometry it must be liquid form in tube reaction. Then,
DNS reagent given just for give the color to easier tested and checked.
Based on Lehninger and Sastrohamidjodjo, DNS is an aromatic compound that
will react with reducing sugars and other reducing components to form 3-amino-5-
nitrosalicylic acid, a compound capable of strongly absorbing electromagnetic wave
radiation at 540 nm. The more reducing components contained in the sample, the more 3-
amino-5-nitrosalicylic acid molecules are formed and the higher the absorption. The
reaction with DNS that occurs is a redox reaction in the sugar aldehyde group and is
oxidized to the carboxyl group. Meanwhile DNS as an oxidizer will be reduced to form
3-amino and 5-nitrosalicylic acid. This reaction runs in an alkaline atmosphere. If there is
reduced sugar in the sample, then the DNS solution that is initially yellow will react with
reducing sugars, giving rise to a reddish orange color.
In making DNS reagents, we need to add NaOH to the solution which aims to
provide an alkaline atmosphere. Because later the reaction of the DNS reagent works in
an alkaline atmosphere. In addition to adding NaOH, also added potassium sodium
tartrate 40% (Rochelle Salt). The function of this addition is to stabilize the color formed
when the reaction occurs namely brick / brownish. In addition, it is also sometimes
necessary to warm up to help speed up the reaction. Because later what will be measured
is the absorbance of the color formed by spectrophotometry at a wavelength of 575 nm.
 After all the sample have been ready, they will enter spectrophotometry. This
practicum used the manual one not the modern one. So first, we must connect the
spectrophotometry into electricity. Then, turn the knob to set the wavelength to 540 nm.
Turn the other knob to convert the spectrophotometer starting at 0. Therefore, we need
blanko (the same solution without glucose) for covert it. After all ready, start to put the
samples one by one. But, always interspersed by entering the blanko as well. We must
note the number that measured. So, actually what happen inside the spectrophotometry is
the tool will shoot light it could be UV or infrared or else into the tube reaction with the
wavelength that had arranged. The number that noted means how much light can pierce
the solution as we know, we put the glucose with different concentrations. Based on
Germer, Spectrophotometry is the quantitative measurement of the interaction of
ultraviolet (UV), visible, and infrared radiation. The nature of this interaction depends
upon the physical properties of the material, for example, transparent or opaque, smooth
or rough, pure or contaminated, and thin or thick.
As we see in the result, there are table from the samples that we made and the
table from example that had been made before. The table that we made is actually passed
several measurements in spectrophotometry. So, there are many numbers and we take the
average. Actually, the right one is on the example table. It should, the smaller the
concentration the more transmittance and the greater the absorbance rate. The result is
different it might be our mistaken before like when measured the weight of solid glucose
or when entered the solid glucose into tube reaction there are fallen glucose.

VI. Conclusion
To measure the carbohydrate quantitatively, there are many ways but one of that
is using Spectrophotometry. Spectrophotometry is a tool that use light for the principal
work. Then, the result we can get standard curve with the formula: Y = aX + b (X =
concentration of reducing sugar, Y = absorbance axis) Also, we can know, the smaller the
concentration of glucose the more transmittance and the greater the absorbance rate.
REFERENCE

Germer, T.A., Zwinkels, J.C. and Tsai, B.K., 2014. Spectrophotometry: Accurate measurement
of optical properties of materials (Vol. 46). Elsevier.

Human Metabolome Database. D-Glucose. http://www.hmdb.ca/metabolites/HMDB0000122

(accessed June 21st, 2019)

Lehninger, A.L. 1997. Dasar-dasar Biokimia (edisi ke-Jilid 1, diterjemahkan oleh M.

Thenawidjaja). Jakarta: Erlangga.

Nielsen, S.S. ed., 2010. Food analysis (pp. 139-141). New York: Springer.

Sastrohamidjojo, Hardjono. 2005. Kimia Organic, Sterokimia, Lemak, dan Protein.

Yogyakarta: Gadjah Mada University Press.

Shendurse A.M., and Khedkar C.D. 2016. Glucose: Properties and Analysis. In: Caballero, B.,
Finglas, P., and Toldrá, F. (eds.) The Encyclopedia of Food and Health vol. 3, pp. 239-
247. Oxford: Academic Press.