1.

0 Abstract

Carbohydrate is one of the main classes of biomolecules. Carbohydrates are varying of

compounds including sugars, starches, celluloses, and fibre. The total carbohydrate is all of
these types added together. Carbohydrate is organic compounds that acting as source of
energy for metabolic reaction. The value of total carbohydrate may vary in each type of
carbohydrate. The objectives of this experiment are to tabulate data for the standard curve
and to estimate the total concentration carbohydrate contain in each sample. The
experiment is conducted by using DNSA reagent which detects reducing ends of
carbohydrate. We also used DNSA reagent in order to determine the total carbohydrate in
each sample. In the end of the experiment, it was observed that the carbohydrate containing
reducing end are fructose, maltose and glucose. It can be detected in DNSA assays. As in
DNSA assays, it is used to calculate the total carbohydrate contained in each sample of
carbohydrate.

2.0 Introduction

Carbohydrates are one of the four major classes of biomolecules along with lipid, nucleic
acids and protein. Their extensive roles make them important as it will make up most of the
organic matter in our surroundings. (M.Berg, 2001) The earlier research about
carbohydrates is about 1940s and 1950s.It has been done by a group of researcher which is
known as The Northern Regional Research Laboratory (NRRL). Start doing research about
new uses of corn, wheat and agricultural waste materials. NRRL solely focused on
carbohydrates because it was a major constituent of the locally grown and corn is the
complex carbohydrates known as starch. Their objectives are to improve the conversion of
starch to glucose and it could be converted to fuel alcohol more cheaply and efficiently.
(L.Cote, 2008) Related to this experiments, we are about to go deeply on carbohydrates and
its type. We will find the unknown concentration and unknown type of carbohydrates by
comparing the results. In this experiment, we will find the most important structure consist in
each of the carbohydrates that will affected the readings of spectrophotometre as well.

3.0 Objective

 To differentiate between reducing and non-reducing sugars. This method detect the
presence of free carbonyl group (C=O), which are the part of the reducing sugars.
 To identify unknown concentration of carbohydrates by mean of standard curve.

1
4.0 Theory

Carbohydrates are classified as organic compound that usually accommodate the source for
metabolic energy to organism and provide structural material such as cellulose (Miloski,
Wallace, Fenger, Schneider, & Bendinskas, 2008), recognition place on cell surfaces and
crucial part for the components of DNA (Deoxyribonucleic acid) and RNA (Ribonucleic acid)
(mb@himedialabs.com). The simplest carbohydrate units is known as monosaccharides or
simply called as simple sugars. Condensation reaction of two monosaccharides will
eventually cause the glycosidic bond to form between of them and this will result a unit of
disaccharides. Meanwhile more than two monosaccharides that involve in condensation
reaction will produce a polysaccharide. However, disaccharide can be broken down into two
units of monosaccharides by hydrolysis reaction. (Miloski, Wallace, Fenger, Schneider, &
Bendinskas, 2008).

Reducing and non-reducing sugar is the major division under carbohydrates. Theoretically,
most researchers have proven that sucrose and starch are categorized as non-reducing
sugar meanwhile glucose, fructose, maltose and lactose are categorized under reducing
sugar based on experiments that have been conducted. Basically, reducing sugar has free
aldehyde and ketone groups in the molecules of sugar. In this experiment, the DNSA (3,5-
dinitrosalicylic acid ) reagent has been used to discover reducing end of carbohydrates. The
free carbonyl group (C=O) of reducing sugar can be detected by using DNSA reagent. This
can be done by oxidation of aldehyde functional group that take part in glucose and ketone
functional group that take part in fructose. Under alkaline solution which is sodium hydroxide
and absorbance of 540nm, the DNSA is reduced to 3- amino-5-nitrosalicylic acid (ANSA)
which turned to a reddish brown coloured complex. (mb@himedialabs.com)

COOH OH COOH OH

+ 6H+ + 6e + 2H2O
NO2 NH2

NO2 NO2

3, 5 Dinitrosalicylic acid (DNSA) 3- amino- 5 nitrosalicylic acid (ANSA)

Figure 4.1: DNSA method of reaction.

2
The equipment that has been used to measure absorbance in this case is
spectrophotometer. This can be done by putting the sample inside a cuvette. The one side of
cuvette is directed with light of certain wavelength and the intensity of light that passed
through the detector is measured. The intensity colour of the product is based on the
concentration of reducing sugar. These explain why the ANSA product appeared as orange
in colour. A calibration curve of standard glucose and fructose should be straight line (Miller,
1959). This experiment involves the calibration curve of absorbance at 540nm versus
concentration of sugar. The law that correlated to this matter is called well-known as Beer-
Lambert Law.

The equation of Beer-Lambert’s law is A=ɛlc .

A is absorbance

ɛ represents molar absorptivity

l is path length

c is concentration of solution.

This equation briefly states that the longer the path length, the lights need to pass through a
lot more of solution, hit more molecules and be absorbed. Thus, this will cause the
absorbance to increase and making the solution to appear darker than original as less light
can be transmitted through it. The same way goes to concentration. If there is increase in
concentration solution, the more molecules of light that is need to pass through. A large
number of ɛ gives a steep gradient hence reflects an indication of strong absorbance.
(Burgess, C.,Mielenz, K.D., 2012).

5.0 Material and apparatus

 Carbohydrates (glucose, fructose, sucrose, maltose and starch)

 3,5-dinitrosalicylic acid (DNSA)
 Rochelle salt; potassium sodium tartrate tetrahydrate
 0.4 M sodium hydroxide, NaOH
 Spectrophotometer
 Beaker (with various sizes)
 Micropipette

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 Laboratory weight scale
 Cuvette
 Magnetic stirrer
 Universal bottle
 Laboratory water bath

Figure 5.1: Laboratory water bath

Figure 5.2:
Spectrophotometer

Figure 5.3: Line of universal bottles filled with

samples

Figure 5.4: Students was using micropipette

4
6.0 Procedures

1) Preparation of carbohydrates
i. There are five types of carbohydrates (glucose, sucrose, maltose and starch)
that are in powdery state need to be prepared by diluting them with distilled
water.
ii. Calculations were made to determine the exact amount of distilled water and
carbohydrates to be mixed in order to achieve desirable concentration (1.4M
to 0.4M and 0.2M).
iii. Then the diluted carbohydrates are used for serial dilution. This will later on
be used for standard curve with DNSA reagent.
2) Preparation of DNSA reagent
i. 20 mL of 0.4M NaOH was dissolved with 1 g of DNSA and 50 mL of distilled
water.
ii. Then, 30 g of Rochelle salt was added.
iii. By the use of distilled water, the mixture’s volume was brought up to 100 mL.
3) Analysis using DNSA reagent
i. 0.8 mL of DNSA reagent and 0.8 mL of sample (all range of serial dilution of
carbohydrates done before this; from 1.4M to 0.2M) was mixed and heated in
boiling water bath for about 10 minutes.
ii. The mixture then diluted with 4 mL of water to give practical amount for
absorbance determination. Absorbance is determined at 540 nm. The types of
various carbohydrates were determined by its colour change.
iii. To use the spectrophotometer, first the absorbance value must read as 0 by
having distilled water (blank adjust use distilled water because it’s the solvent
used to dissolved sample) contained in a cuvette, put into chamber.
iv. Only after zero absorbance was read, the absorbance of mixture of first
concentration was read.
v. The (iii) and (iv) steps were repeated with numbers of concentration of
carbohydrates.
vi. Result was taken and standard curve was drawn.

5
7.0 Results

Graph of Absorbance vs Molarity

0.05

0.045

0.04 R² = 0.0874

0.035

0.03
ABSORBANCES

0.025

0.02

0.015

0.01

0.005

0
-6 -5 -4 -3 -2 -1 0 1 2
MOLARITY(MOL/L)

Figure 7.1: Graph of absorbance against molarity of glucose

6
 Table 7.1: Data of absorbances and molarity of glucose

Absorbances

1 2 3 Average

Molarity(moles/L)

Standard 0.041 0.038 0.038 0.039

1.2 0.038 0.038 0.039 0.038

1.0 0.033 0.032 0.035 0.033

0.8 0.045 0.043 0.043 0.044

0.6 0.027 0.026 0.028 0.027

0.4 0.039 0.037 0.036 0.037

Table 7.2: Data of absorbances for solution D.

Trial Readings of absorbances

1 0.015
2 0.002
Average = 0.009

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 Graph of Absorbance vs Molarity.
0.05

0.045

0.04 R² = 0.306
0.035
ABSORBANCES

0.03

0.025

0.02

0.015

0.01

0.005

0
-2 -1.5 -1 -0.5 0 0.5 1 1.5 2
MOLARITY(MOL/L)

Figure 7.2: Graph of absorbance against molarity of sucrose

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 Table 7.3: Data of absorbances and molarity of sucrose

Absorbance
s
1 2 3 Average

Molarity(moles/L
)
Standard 0.029 0.030 0.029 0.029

1.2 0.045 0.045 0.045 0.045

1.0 0.036 0.036 0.035 0.036

0.8 0.028 0.030 0.030 0.029

0.6 0.028 0.029 0.018 0.025

0.4 0.026 0.028 0.028 0.027

Table 7.4: Data of absorbances for solution C

Trial Readings of Absorbances

1 0.016
2 0.003
3 0.015
Average = 0.011

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 Graph of Absorbance vs Molarity.
0.03

0.02
R² = 0.0798
0.01

0
ABSORBANCES

0 0.5 1 1.5 2 2.5 3

-0.01

-0.02

-0.03

-0.04

-0.05

-0.06

-0.07
MOLARITY (MOL/L)

Figure 7.3: Graph of absorbance against molarity of maltose

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 Table 7.5: Data of absorbances and molarity of maltose

Absorbance
s
1 2 Average

Molarity(moles/L
)
Standard -0.015 -0.025 -0.02

1.2 0.021 0.015 0.018

1.0 -0.076 -0.052 -0.064

0.8 -0.033 -0.033 -0.033

0.6 -0.051 -0.053 -0.052

0.4 -0.022 -0.008 -0.015

Table 7.6: Data of absorbances of solution A

Trial Readings of Absorbances

1 0.001
2 0.000
3 0.000
Average = 0.001

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 Graph of Absorbance vs Molarity
0.8

0.6
R² = 0.2415

0.4
ABSORBANCE

0.2

0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
-0.2

-0.4

-0.6
MOLARITY (MOL/L

Figure 7.4: Graph of absorbance against molarity of starch

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 Table 7.7: Data of absorbances and molarity of starch

Molarity(moles/L) Absorbances

Standard -0.068

1.2 0.408

1.0 0.676

0.8 0.564

0.6 0.1825

0.4 -0.067

Table 7.8: Data of absorbances of solution B

Trial Readings of Absorbances

1 0.001
2 0.000
3 0.000
Average = 0.001

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8.0 Calculations

Molecular weight for:

1. Glucose: 180.1559 g/mol

2. Sucrose: 342.2965 g/mol
3. Maltose: 342.3 g/mol
4. Starch: 162.16 g/mol

Number of moles (mol)

Molarity (M) =
Volume (L)

Number of moles (mol) = Molarity(M) × Volume(L)

g
Mass (g) = Number of moles (mol) × Molecular weight ( )
mol

Mass for preparation of standard dilution:

Type of carbohydrate Mass (g)

Glucose (1.4M x 0.025L) x (180.1559 g/mol) = 6.31
Sucrose (1.4M x 0.010L) x (342.2965 g/mol) = 4.79
Maltose (1.4M x 0.010L) x (342.3 g/mol) = 4.79
Starch (1.4M x 0.010L) x (162.16 g/mol) = 2.27

Formula for serial dilution:

M1V1 =M2V2

Serial dilution for:

1. Glucose
(1.4 mol/L) x V1 = (1.2 mol/L) x (0.025L)
V1 = 0.0215L + 0.0035L
*0.0035L of water must be added

(1.2 mol/L) x V1 = (1.0 mol/L) x (0.025L)

V1 = 0.02075L + 0.00425L

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 *0.00425L of water must be added

(1.0 mol/L) x V1 = (0.8 mol/L) x (0.025L)

V1 = 0.02L + 0.005L
*0.005L of water must be added

(0.8mol/L) x V1 = (0.6 mol/L) x (0.025L)

V1 = 0.01875L + 0.00625L
*0.00625L of water must be added

(0.6mol/L) x V1 = (0.4 mol/L) x (0.025L)

V1 = 0.0175L + 0.0075L
*0.0075L of water must be added

2. Sucrose
(1.4 mol/L) x V1 = (1.2 mol/L) x (0.010L)
V1 = 0.00857L + 0.00143L
*0.00143L of water must be added

(1.2 mol/L) x V1 = (1.0 mol/L) x (0.010L)

V1 = 0.0083L + 0.0017L
*0.0017L of water must be added

(1.0 mol/L) x V1 = (0.8 mol/L) x (0.010L)

V1 = 0.008L + 0.002L
*0.002L of water must be added

(0.8mol/L) x V1 = (0.6 mol/L) x (0.010L)

V1 = 0.0075L + 0.0025L
*0.0025L of water must be added

(0.6mol/L) x V1 = (0.4 mol/L) x (0.010L)

V1 = 0.00667L + 0.00333L
*0.00333L of water must be added

3. Maltose
(1.4 mol/L) x V1 = (1.2 mol/L) x (0.010L)

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 V1 = 0.00857L + 0.00143L
*0.00143L of water must be added

(1.2 mol/L) x V1 = (1.0 mol/L) x (0.010L)

V1 = 0.0083L + 0.0017L
*0.0017L of water must be added

(1.0mol/L) x V1 = (0.8 mol/L) x (0.010L)

V1 = 0.008L + 0.002L)

*0.002L of water must be added

(0.8mol/L) x V1 = (0.6 mol/L) x (0.010L)

V1 = 0.0075L + 0.0025L

*0.0025L of water must be added

(0.6mol/L) x V1 = (0.4 mol/L) x (0.010L)

V1 = 0.00667L + 0.00333L

*0.00333L of water must be added

4. Starch
(1.4 mol/L) x V1 = (1.2 mol/L) x (0.010L)

V1 = 0.00857L + 0.00143L

*0.00143L of water must be added

(1.2 mol/L) x V1 = (1.0 mol/L) x (0.010L)

V1 = 0.0083L + 0.0017L

*0.0017L of water must be added

(1.0 mol/L) x V1 = (0.8 mol/L) x (0.010L)

V1 = 0.008L + 0.002L

*0.002L of water must be added

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 (0.8mol/L) x V1 = (0.6 mol/L) x (0.010L)

V1 = 0.0075L + 0.0025L

*0.0025L of water must be added

(0.6mol/L) x V1 = (0.4 mol/L) x (0.010L)

V1 = 0.00667L + 0.00333L

*0.00333L of water must be added

Sample Of Calculation Error

In order to get the best fit line, the value of R2 must be 0.9.

Percentage error = (approximate value – exact value) x 100%

Percentage error of R2 for:

1. Glucose = (0.0874-0.9) x 100%

= 81%

2. Sucrose = (0.306-0.9) x 100%

= 59%

3. Maltose = (0.0798-0.9) x 100%

= 82%

4. Starch = (0.2415-0.9) x 100%

= 66%

9.0 Discussion

Glucose

Based on the result, the colour that presents from glucose solution is yellow-orange colour.
So, we can conclude that glucose is a reducing sugar. Reducing sugar is a type of
carbohydrates that undergo oxidation by weak oxidizing agent in basic aqueous solution.
The features that carry out by reducing sugar are they have an open chain with an aldehyde
or a ketone group. Based on the graph in Figure 7.1, the R2 value stated for glucose is
0.0874 which is farther from the value 1.0. Theoretically, the closer the value of R2 towards

17
1.0, the better the fit of the regression line. The farther value probably due to error during the
experiments for examples, the cuvettes does not clean properly before read the absorbance.

For section C of the experiments, we can predict the unknown type of sugar by referring to
the colour presents by the solution. In this experiments, we can conclude that for solution D,
the colour appeared is yellow-orange colour gives its as a glucose solution. For its unknown
concentration, we can directly get from graph by plotting the value of absorbance on the
graph. Here we get the concentrations of the solutions is -0.482. Basically, there is no
negative value for concentration. But in this case, the negative value probably by the error
during the experiments such as the sample has been contaminated yet the sample become
unstable and produce less accurate of absorbance readings.

Sucrose

Sucrose is categorized under non-reducing sugar based on this experiment. This is because
the colour appeared after the sample is mixed with DNSA reagent and boiled in the water
bath is yellow in colour. As mention earlier in the theory of this experiment, the yellow in
colour is categorized as non-reducing sugar meanwhile orange-yellow in colour is
categorized as reducing sugar. This is based on the structure of sucrose itself as it does not
have free carbonyl group that is essential for reaction. In sucrose, the reducing end of the
component monosaccharides are separated by glycosidic bond hence it does the ability to
react with DNSA. According to the graph, the value of r2 for sucrose is 0.306. In this context
of discussion, the closer value of r2 to 1.0, the regression of line is much better. Based on the
experiment, we can consider that 0.306 is much farther from 1.0 which is due to the errors
present during the implementation of the experiment. Most probably the error comes from
unclean cuvette hence it disturbs the accuracy reading of spectrophotometer absorbance.
For determining the unknown concentration of sugar, it is crucial to observe the colour of the
unknown and using the graph of absorbance versus concentration to find the concentration
of given absorbance. From the experiment, unknown C is concluded as sucrose as it yellow
in colour. Meanwhile, the concentration of sucrose is -1.15M. This is obviously contradicted
to the nature of concentration as it cannot present in negative value. Again, this might due to
the unclean cuvettes, wrong position of cuvettes in spectrophotometer or the device itself
may not functionally well.

Starch

At first, for preparing standard curve (absorbance vs. concentration of carbohydrates), we

need to determine the precise amount of water and carbohydrates that need to be diluted.
Calculation was done according to the formula relating concentration and volume. Then, the

18
starch solution along with its serially diluted versions was taken for measurement of their
absorbance by using spectrophotometer. In before, the DNSA reagent was prepared. It’ll
then in use for detection of absorbance.
DNS binds only to reducing sugars and cannot bind to non-reducing sugars (e.g.: starch)
because it only detects reducing ends of carbohydrate (Miloski, Wallace, Fenger, &
Schneider, 2008). Starch is a polysaccharide, it’s a non-reducing sugar because its chemical
formula is stable and it does not oxidize. During the incubation period, enzyme acts upon
substrates to give products which are reducing sugars. Since starch is non-reducing sugars,
the colour stays as yellow and 3,5-dinitrosalicylic acid, DNS, did not change to 3-amino, 5-
nitrosalicylic acid, ANS. Changing of mixture colour caused by the presence of free carbonyl
group (C=O), the so-called reducing sugars.
Absorbance is directly proportional to the concentration of the absorbing species
(Albalasmeh, Berhe, & Ghezzehei, 2013). The intensity of the colour is directly proportional
to the amount of reducing sugar present in the sample. This intensity change in colour
(which it’ll get darker if there’s more free carbonyl group presence) is measured using a
colorimeter (or as in this experiment, using a spectrophotometry) as the absorbance set at
540nm wavelength. The correlation coefficient (R) was calculated by the least squares
method; used to indicate the adequacy of the curve to the mathematical model (Negrulescu,
Patrulea, Mincea, Ionascu, Vlad-Oros, & Ostafe, 2012).
The absorbance reading for starch is not in a constant value, which it have both negative
and positive reading at some concentration. However, negative absorbance has no physical
meaning except the fact that the blank absorbs more light than the sample, meaning that the
solution could be too dilute, experiment conducted with contaminated/empty cuvette, or
calibration of machine did not happened using blank. Beer-Lambert’s Law state that
absorbance and concentration of an absorbing species have a linear relationship between
them. The absorbance at maximum is equal to 1, and 0 means there is 100% transmittance,
(all the light has pass through). According to Beer's law, the absorbance of a solution should
be zero (100%T) if there is none of the absorbing species present.

Meanwhile, for the part (c), the unknown concentration and solution can be known by
comparing data available from part (a); which is the standard curve. However the negative
concentration could be obtained if there’s almost no glucose (starch) available in the
samples (Streb, 2015).

Maltose

Maltose is commonly called as malt sugar or also known as crystalline disaccharide which
made of two glucose monomers subunits (htt5). Its empirical formula (C12H22O11) is similar

19
with lactose and sucrose but its structure varies from both (htt5). In fact, maltose is a
reducing sugar. That’s mean maltose is a type of carbohydrates which contain free aldehyde
or keto group. In this experiment, we noticed that maltose can also be used as a standard in
order to estimate reducing sugar in unknown samples. By constructing graph for maltose, we
can estimate molarity (M) of reducing sugars present in unknown samples. The result for
maltose is the colour of pale yellow DNSA turned to orange-red colour. Beer Lambert’s Law
stated that this colour intensity is proportional to the molarity of maltose present in the
solution (htt6). This colour intensity change is then measured by using the
spectrophotometer. The reason why it changes colour is because 3,5-dinitrosalicylic acid
reacts with maltose to form 3-amino,5-nitrosalicylic acid in a reduction reaction (Biology-
Forums.com). The chemical formed is then is able to absorb light at 540 nm (Biology-
Forums.com).

For part C which is analysis using DNSA reagent, we need to use dilutions of a solution of
known concentration to determine concentration of unknown. After observed the colour
change of Maltose, we can conclude that maltose showed positive result that means it is a
reducing sugar. The absorbance for this reducing sugar supposedly has range more than
1.0. However, from the graph (data collected) we got negative value for maltose. This
showed that there are errors occurred. The possible error is the ways we handled pipette is
wrong such as we are not cleaning the pipette with distilled water correctly. Pipetting
technique is the most important to have accurate data and also to prevent contamination of
samples (htt5). Other possibility is the cuvette is not handled properly.

10.0 Conclusion

By the end of the experiment, we can learn the technique of spectrophotometric

carbohydrate assays. The dinitrosalicylic acid (DNSA) has been used in this experiment in
order to identify the reducing ends of carbohydrates. The standard curve for glucose,
sucrose, maltose, and starch has been done. Then, we used dilution of solution of known
concentration to determine unknown concentration in part c. The slope of this graph (partA)
was used to determine the concentration in unknown samples. From this experiment, we can
say that the higher the sugar content, the higher the carbonyl group and the darker it
become (reducing sugar). We can also conclude that colour intensity is proportional to the
molarity of given type of carbohydrate present in the solution. Unfortunately, the results that
we got seemed to have some errors. For example, the result for maltose which is a reducing
sugar supposed to have the absorbance value in range over 1 but we got negative value

20
instead. However, the color of maltose showed the positive result means that it is reducing
sugar. The possibilities of errors have been stated in the discussion.

11.0 Recommendations

i. Apart from stick to only using DNS method, we can try few other alternatives such as
Nelson-Somogyi’s method (but it cannot be used for sucrose) and ortho-toluidine
method (DNS assay is known to be approximately 10 times less sensitive than the
NS assay). Result obtained from various methods can be compared to determine the
most accurate end result.
ii. Adding supplemental cellobiase can eliminate cellobiase inhibition thus reducing
error when measuring the total reducing sugars on the DNSA assay.
iii. Increasing the boiling time (only up to 15 minutes) can help to ensure the colour
development reaction completely occurs.
iv. Add phenol to increase the intensity of colour produced (must add sodium bisulfite
together to stabilize the colour obtained). Absence of bisulfite resulted in inability to
indicate tiny amount of amylase especially with starch substrates.

12.0 References

Albalasmeh, A. A., Berhe, A. A., & Ghezzehei, T. A. (2013). A new method for rapid
determination of carbohydrate and totalcarbon concentrations using UV
spectrophotometry. Carbohydrate Polymers 97 , 253-261.

Berg, J. M., Tymoczko, J. L., & Stryer, L. (2012). Biochemistry (7th ed.). Houndmills,
England: W. H. Freeman & Palgrave Macmillan.

Miloski, K., Wallace, K., Fenger, A., & Schneider, E. (2008). Comparison of Biochemical and
Chemical Digestion and Detection Methods for Carbohydrates. American Journal of
Undergraduate Research Vol. 7, No. 2, 7-18.

Negrulescu, A., Patrulea, V., Mincea, M. M., Ionascu, C., Vlad-Oros, B. A., & Ostafe, V.
(2012). Adapting the Reducing Sugars Method with Dinitrosalicylic Acid to Microtiter
Plates and Microwave Heating. J. Braz. Chem. Soc., Vol. 23, No. 12, 2176-2182.

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Streb, S. (23 February, 2015). How do you measure the glucose concentration of an
unknown sample? Retrieved 12 October, 2015, from ResearchGate:
http://www.researchgate.net/post/How_do_you_measure_the_glucose_concentration
_of_an_unknown_sample

L.Cote, G. (2008). A History of Carbohydrate Research at the USDA Laboratory in Peoria.

103-105.

(n.d.). Retrieved from

http://life.nthu.edu.tw/~labcjw/BioPhyChem/Spectroscopy/beerslaw.htm

Biology-Forums.com. (n.d.). Retrieved from http://biology-

forums.com/index.php?topic=1360.0

HiPer® Carbohydrates Estimation Teaching Kit (Quantitative). (n.d.). Retrieved October 15,
2015, from http://himedialabs.com/TD/HTBC003.pdf

Burgess, C., & Mielenz, K. D. (Eds.). (2012). Advances in Standards and Methodology in
Spectrophotometry. Elsevier.

Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing

sugar. Analytical chemistry, 31(3), 426-428.

Bakeev, K. (2010). Process analytical technology spectroscopic tools and implementation

strategies for the chemical and pharmaceutical industries (2nd ed.). Chichester, West
Sussex: Wiley.

22
13.0 Appendices

Figure 13.1: DNS measurement

Figure 13.2: Laboratory weight scale

23
Appendix B

24
25
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