MOLLISCH TEST

The Molisch test is a chemical test used to detect the presence of carbohydrates,
particularly the presence of any form of carbohydrates containing more than three
carbon atoms. This test is based on the ability of carbohydrates to form a violet or
purple color in the presence of concentrated sulfuric acid and alpha-naphthol.
PRINCIPLE

MATERIALS:
1) Sample containing the carbohydrate to be tested
2) α-naphthol solution (5% w/v in ethanol)
3) Concentrated sulfuric acid
Procedure:
1) Prepare the Sample:Ensure your sample is in a liquid state. If it's a solid,
dissolve it in water or an appropriate solvent.
2) Filter the solution if necessary to remove any insoluble particles.
3) Molisch Reagent Preparation:Prepare the α-naphthol solution by dissolving
α-naphthol in ethanol to make a 5% w/v solution.
4) Take a small volume (usually 2-3 drops) of the prepared sample solution in a
test tube.
5) Add a few drops of Molisch reagent (α-naphthol solution) to the sample.
Mix well.
6) Sulfuric Acid Addition:
7) Incline the test tube and carefully add concentrated sulfuric acid down the
side of the tube so that it forms a layer at the bottom without mixing with the
sample. You can do this by placing the tip of a dropper against the side of
the tube.
Observation:
Observe the appearance of a violet or purple ring at the interface of the two layers.
The color development indicates the presence of carbohydrates.
Note:
 A positive test result is the formation of a violet or purple color within a few
minutes.
  The Molisch test is a sensitive test for the presence of carbohydrates but is
not specific for a particular type of carbohydrate.
 It's important to handle concentrated sulfuric acid with care due to its
corrosive nature. Perform the test in a well-ventilated area and use
appropriate safety precautions.

You
principle of the above test
ChatGPT
ChatGPT
The Molisch test is based on the principle of dehydration and condensation
reactions. The test detects the presence of carbohydrates, including
monosaccharides, disaccharides, and polysaccharide. The α-naphthol solution
reacts with carbohydrates in the sample. Carbohydrates are polyhydroxy
compounds, meaning they contain multiple hydroxyl (-OH) groups.The α-naphthol
reacts with these hydroxyl groups to form a complex mixture of compounds.
The addition of concentrated sulfuric acid to the mixture results in dehydration and
condensation reactions. Sulfuric acid acts as a dehydrating agent, removing water
molecules from the complex formed between α-naphthol and carbohydrates.
This leads to the formation of furfural or its derivatives from the carbohydrates
present in the sample.
Furfural or its derivatives react further with α-naphthol to produce a colored
compound.
The appearance of a violet or purple color at the interface of the two layers
indicates a positive result for the presence of carbohydrates in the sample.
In summary, the Molisch test relies on the ability of carbohydrates to react with α-
naphthol, and subsequent dehydration and condensation reactions with sulfuric
acid lead to the formation of a colored product. The test is sensitive to the presence
of carbohydrates but is not specific for a particular type of carbohydrate. It's a
general test used for the qualitative detection of carbohydrates in a given sample.
You
benedicts test
ChatGPT
ChatGPT
The Benedict's test is a chemical test used to detect the presence of reducing sugars
in a given sample. Reducing sugars are carbohydrates that have free aldehyde or
ketone functional groups, which can reduce certain metal ions, such as copper ions
(Cu^2+), to form a colored precipitate. The test is commonly used to detect the
presence of sugars like glucose and maltose. Here's a general procedure for the
Benedict's test:
Materials:
Sample containing the reducing sugar to be tested
Benedict's reagent (solution containing copper sulfate, sodium citrate, and sodium
carbonate)
Procedure:
Prepare the Sample:
Ensure your sample is in a liquid state. If it's a solid, dissolve it in water or an
appropriate solvent.
Benedict's Reagent Preparation:
Benedict's reagent is usually prepared by dissolving copper sulfate, sodium citrate,
and sodium carbonate in water. The specific composition may vary, but a common
formula includes 0.1 M copper sulfate, 1% sodium citrate, and 2% sodium
carbonate.
Benedict's Test Reaction:
Take a small volume (usually 2-3 mL) of the prepared sample solution in a test
tube.
Add Benedict's Reagent:
Add an equal volume of Benedict's reagent to the sample solution.
Heat the Mixture:
Heat the test tube containing the mixture in a boiling water bath or gently over an
open flame for a few minutes. The heating step is crucial for the reaction to occur.
Observation:
Observe any color change in the reaction mixture. The development of a colored
precipitate (typically a brick-red or orange precipitate) indicates the presence of
reducing sugars.
Note:
The color change is due to the reduction of copper ions in the Benedict's reagent by
the reducing sugars. The intensity of the color can vary depending on the
concentration of reducing sugars in the sample.
Benedict's test is not specific for a particular type of reducing sugar and can also
give positive results with other reducing substances.
Top of Form
It should not be done in a person with confirmed Diabetes mellitus
patients that is Fasting blood glucose level more than 126 – 140 mg/dl
and Postprandial glucose level more than 200 mg/dl.

Clinical Significance
In normal person, the fasting plasma glucose level is 70 – 110 mg/dl.
Following glucose ingestion, the level rises and reaches the peak with in
half or one hour. The plasma glucose level then returns rapidly to the
fasting normal level at 2½ hours. This is due to the secretion of insulin in
response to the elevation of blood glucose. None of the urine sample
shows any evidence of glucose.

Diminished glucose tolerance is present when ability of the body to

utilize glucose decreases. It is observed in diabetes mellitus. The rise in
plasma glucose is greater than the normal persons and return to plasma
glucose after 2½ hours is delayed. Similar curve also observed in case of
hyper activities of thyroid, pituitary and adrenals, injection of
adrenocorticotropic hormone, increased secretion of Growth hormone,
severe liver diseases, severe infection etc.
Lag type of tolerance curve observed, when temporary greater increase
of plasma glucose after ingestion of glucose is due to delay insulin
mechanism action. This is due to increase absorption of glucose from the
intestine or delay in the insulin mechanism coming into action. This may
occur following period deficient of carbohydrate intake and also a
patient after gastractomy, peptic ulcer, hyperthyroidism and hepatic
disease etc. In case of alimentary Diabetes mellitus transient glycosuria
may occur.

Increase glucose tolerance that is the ability of the body to utilize the
glucose is increase. This is observed in case of hypo activity of thyroid,
pituitary and adrenals. Some time, the fasting blood glucose level goes
to below normal limit. Similar GTT curve observe in patient with
idiopathic steatorrhoea.

Preparation of patients
 The patient should be on balanced diet (containing normal daily
requirement of carbohydrates) at least for 2 – 3 days prior to the test.
 All drugs known to influence carbohydrate metabolism should be
discontinued for at least 2 days prior to the test.
 The patients should avoid strenuous exercise on the previous day of
the test.
 The patient should report to the laboratory after fasting for 12 – 16
hours. He/She can drink water.
 He/She should bring fasting midstream urine sample collected in a
clean and dry container.
 During the course of GTT, the person should be comfortably seated
and should abstaining smoking and exercise.
 The patient should be waiting in the laboratory for at least 2 – 3 hours,
since five blood sample and urine sample are collected at the interval
of 30 minutes.

Procedure of GTT
Glucose tolerance test should be conducted preferably in the morning
(ideal time : 9 am –11 am).

1. Collected fasting blood sample and also morning midstream

urine specimen (called ‘O’ hour sample).
2. Given 75 -100 mg of anhydrous glucose (1.75 gm/Kg weight)
dissolved in 250-300 ml of water to the patient and told the patient to
drink slowly with in about 5 minutes. Note the time.
3. Collected four more blood sample at the 30 minutes intervals for two
hours after the glucose has been taken.
4. Four urine samples are collected after the collection of each blood
samples (if the patient unable to give four urine samples, collected at
least two urine samples at the one hour interval)
5. Glucose is estimated in all the blood samples and urine samples are
tested for glucose qualitatively by using suitable methods.
6. Prepared a GTT curve by plotting Time on X-axis and Plasma glucose
on Y-axis and give the result according to the GTT curve.

Interpretation of GTT
The graphic representation of the GTT result is depicted in the fig. GTT
curve.

Normal response
In normal person the fasting plasma glucose level is 70 – 110 mg/dl.
Following glucose ingestion, the level rises and reaches the peak with in
half or one hour. The plasma glucose level then returns rapidly to the
fasting normal level at 2½ hours. This is due to the secretion of insulin in
response to the elevation of blood glucose. None of the urine sample
shows any evidence of glucose.

In Diabetes Mellitus

1. The fasting plasma glucose level is > 126 mg/dl.

2. Or 2 hours post glucose value is >200 mg/dl.
3. Or both fasting and 2 hour’s values are above this level.
4. Or the random plasma glucose is >200mg/dl.

This is due to deficiency of insulin or others causes such as

hyperthyroidism, hyperpituitarism, hyper activity of adrenals, liver
diseases etc.

Impaired Glucose Tolerance

In case of Impair Glucose Tolerance (IGT), the plasma glucose level is
above normal level and below the diabetic level.

Fasting glucose level : 110 – 126 mg/dl

Post Prandial glucose level : 140 – 200 mg/dl

Such person leads careful follow up because the persons slowly develop
frank diabetes at an estimated rate of 2% per year. Dietary restriction
and exercise are recommended for the treatment of impaired glucose
tolerance.

Gestational Diabetes Mellitus

When carbohydrate intolerance is noticed for a first time during
pregnancy this is known as Gestational Diabetes mellitus (GDM). Here,
fasting glucose level is >126 gm/dl and they are more susceptible for
development of frank diabetes mellitus.


Interpretation of GTT : Time vs Plasma glucose level

Alimentary Diabetes Mellitus

Here, Fasting and Post prandial plasma glucose level are normal
but glucose level rapidly increase after ingestion of glucose and it may
be reach up to renal threshold and afterwards returns to normal limit at
2 hrs. This is due to increase absorption of glucose from the intestine or
delay in the insulin mechanism coming into action. This may occur
following period deficient of carbohydrate intake and also a patient after
gastractomy, peptic ulcer, hyperthyroidism and hepatic disease etc. In
case of alimentary Diabetes mellitus transient glycosuria may occur.

Flat GTT curve

In case of Flat GTT curve, the peak level is usual much lower than the
normal GTT curve. This is due to malabsorption, hypopituitarism
and hypothyroidism etc.

Renal Glycosuria
Renal glycosuria is due to lower renal threshold. Renal threshold in some
persons may be lowered (may be 130 – 150 mg/dl), so that after taking
of glucose (or after a rich carbohydrate meal) when normal glucose
level reached in peak (up to 130 – 150 mg/dl) temporary glycosuria may
take place but the GTT curve is normal. This is observed due to some
abnormality in the tubular re-absorption of glucose.