1
Q
chemical constituents of blood
A
proteins:
- albunim
- globulin
- fibrinogen
- lipids
- glucose
- amino acids
- urea
- uric acid
- creatinine
- hormones
- vitamins
- electrolytes, cations (Na+, K+, Ca2+, Mg2) and anions (Cl-, HCO3-, HPO3-)
2
Q
normal pH of arterial blood
A
7.4
3
Q
what are the 2 things in present and what quantities?
A
45% blood volume → cellular fraction (contains erythrocytes, leukocytes, platelets)
- erythrocytes: red blood cells (carry oxygen)
- leukocytes: white blood cells (help fight infections)
- platelets: help with blood clotting
55% blood volume → plasma (carries nutrients, hormones, and waste products)
4
Q
buffer
A
solution which tends to maintain a constant pH when excess acid or base is added
5
Q
hydrates
A
compounds containing water chemically combined in a definite ratio
- computations using formula weight must take the water molecules into account
6
Q
molality, molar mass, molarity
A
molality: # of moles of solute/# of kilograms of solvent
molar mass: mass of a mole of any element or compound
molarity: # of moles of solute/by liters of solution
7
Q
Q1. what is meant by mole?
A
mole = weight (g)/molecular weight (g/mol)
8
Q
Q2. what is the difference between molecular weight and gram molecular weight?
A
molecular weight of compound → sum of the atomic weight of all the atoms that make up one molecule of the compound
gram molecular weight → molecular weight expressed in grams is termed the gram molecular weight but is often shortened to just “molecular weight”
9
Q
Q3. What is the significance of Molisch’s test?
A
This test is useful for identifying any compound which can be dehydrated to furfural or hydroxymethyl furfural in the presence of H2SO4.
presence of carbs
10
Q
What compounds are formed when pentoses and hexoses get dehydrated by strong mineral acid?
A
Pentoses are dehydrated to furfural
Hexoses are dehydrated to 5-hydroxymethylfulfural
11
Q
Q1. Name non-carbohydrates which also give positive Molisch’s test
A
- Furfurals
- Aldehydes
- Some organic acids (ex. formic acid, oxalic acid. lactic acid. citric acid etc.)
these can also give positive Molisch’s test.
12
Q
Q5. Name the complex formed by the addition of concentrated sulfuric acid to sugar solution ?
A
Furfural (important for detecting presence of carbohydrates)
13
Q
Q6. what are furfurals?
A
key intermediates formed in molich’s test that lead to color change in Molisch’s test
formed when strong acids remove 3 molecules of water from monosaccharides
14
Q
Q4. sometimes a green ring also appears in the bottom of the test tube in Molisch’s test. what is the reason?
A
excessive α-naphthol
(it should be ignored, a lil bit is fine)
15
Q
Q1. What are aldoses?
A
It is a monosaccharide which contains Aldehyde groups.
- ex’s. Glyceraldehydes, Erythrose, Ribose, Glucose. and Glucoheptose
16
Q
Q2. What are ketoses?
A
It is a monosaccharide which contains ketose group.
- ex.’s Dihydroxyacetone.
Erythrulose, Ribulose, Fructose, and Sedoheptulose
17
Q
Q3. Why does cherry red color appear in Seliwanoff’s test?
A
Monosaccharides are resistant to the action of dilute hot mineral acids. Strong acids remove the water and dehydrate hexoses to form Furfurals. When furfurals condense with
phenols they form a colored product.
18
Q
Molisch’s test principle
A
- conc. sulphuric acid hydrolyzes (breaks down) polysaccharides to disaccharides and monosaccharides.
- The monosaccharides are dehydrated by the conc. sulphuric acid to form furfural or one of its derivatives (sulfuric acid removes the water to form furfural).
- Furfural or their derivatives (hydroxymethylfurfural for hexoses) condense with 2 molecules of α-Naphthol (which has iodine) to form violet colored complex.
- Monosaccharides give a rapid positive test.
- Disaccharides and polysaccharides react slower.
19
Q
qualitative vs quantitative tests
A
quantitative detect HOW MUCH is present
qualitative detect whether present or not
20
Q
purpose of Molisch’s test + interpretation (meaning result)
A
qualitatitve (present or absent) test and is a general test for the detection of all types of carbohydrates in a solution (mono, di, poly)
result: reddish violet ring at junction or interface of 2 liquids indicates presence of carbohydrate in the test tube
21
Q
Iodine test principle
A
- Amylose (part of starch) has a coil (helix) shape and also property of adsorption
- Iodine molecules fit inside this coil (adsorption - attach to surface of molecule instead of fully going in)
- The iodine inside the amylose forms a special complex (polyiodine chains) that appear deep blue in the presence of iodine
- if no starch present, then color will remain orange or yellow
22
Q
purpose of iodine test + interpretation (results)
A
purpose: detection of starch (mostly) and glycogen in the given solution, test for polysaccharides (not all- just starch)
interpretation: deep blue color appears that confirms presence of starch in given solution
23
Q
Q1. What do you mean by negative iodine test?
A
If the solution remains the colour of iodine i.e. reddish-orange; this means starch is not present. whereby termed as negative lodine test.
24
Q
Q2. What is the effect of temperature on Iodine test?
A
Heating causes its return to original solution by breaking the colored complex (becomes colorless) and on cooling. the complex is reformed (gains color again).
25
Q3. What is the optimum temperature for accurate iodine test?
At or near room temp i.e. 25ºC
26
Q3. What is the effect of pH on iodine test?
Alkaline medium breaks the complex (NaOH causes it to become colorless) while acidic medium favors its formation (HCl brings color back)
27
Q5. What is the composition of starch?
Natural starches are mixtures of **amylose** (10-20%) and **amylopectin** (80-90%)
28
Q6. What gives deep blue color in Iodine test?
Amylose in starch is responsible for the formation of a deep blue color in the presence of iodine
29
Q7. Which carbohydrates give negative Iodine test?
- Starch amylopectin
- Cellulose
- disaccharides such as sucrose in sugar
(do not give color)
30
Q8. Which factors decrease the intensity of color in Iodine test?
Intensity of the color **decreases with increasing temperature** and with the **presence of water-miscible, organic solvents** such as ethanol.
31
Q9. Why can the iodine test not be done at very low pH?
low pH hydrolyzes (breaks down) the starch
32
Q10. Why is the iodine test most suitable for starch?
Starch in the form of amylose and amylopectin has less branches than glycogen.
Hence the helices of starch are longer than glycogen, therefore **binding more iodine
atoms**.
33
Q11. what is Lugol's iodine?
Solution of elemental iodine and potassium iodide in water
34
Q12. What are the uses of Lugol's iodine?
- antiseptic
- disinfectant
- emergency disinfection of drinking water
- reagent for starch detection in routine lab and medical tests
35
Q. why are monosaccharides soluble in water?
Monosaccharides are quite soluble in water because of the numerous OH groups that readily engage in hydrogen bonding with water
36
principle of **Benedict's Test**
*principle is similar to that of the Fehling's test* i.e. **reducing sugars can reduce cupric ions (Cu2+) to cuprous ions (Cu+) in an alkaline (basic) medium provided by Na2CO3**
1. Reducing sugars reduce ions while oxidizing themselves to sugar acids
2. cuprous ions (Cu+) combined with H2O to form cuprous hydroxide (CuOH)
3. CuOH heated w/ reducing sugars gets converted to cuprous oxide (CuO) - *insoluble in water so forms precipitate of different colors depending upon the concentration of sugar present*
37
**Benedict's test** (type of test + positive result indicator)
**type of test**: simple qualitative & semi-quantitative (different colors represent diff percents) test for **reducing sugars**
percentage of reducing sugars (concentration) present is based on the color!! (can tell up to 2%)
**brick red precipitate appears after a variety of colors which shows the presence of reducing sugars (monosaccharides or disaccharides) - percentage is above 2%**
38
ranges of **Benedict's test** (percentages & colors)
color of precipitate:
**green** → 0.1-0.5%
**yellow** → 0.5-1%
**orange** → 1-1.5%
**red** → 1.5-2%
**above red** → above 2% or high
*why its called semi-quantitative bc can only go up to 2% and cant tell after that*
39
Q. what are reducing sugars? give examples.
All sugars containing a potentially free active aldehyde or keto group i.e. in which
anomeric carbon atom is free and not attached to any other structure are known as
reducing sugars
*ex. all monosaccharides, lactose, maltose*
40
Q. which sugars give a positive test to **benedict's test**?
All monosaccharide & disaccharides will give this test positive **except sucrose & trehalose** because they don't contain free anomeric carbon atom as it is involved in O-glycosidic linkage with other molecules.
41
Q. What is the difference b/w glycosuria & glucosuria?
**Glycosuria**: presence of any reducing sugar in urine (*ex. fructosuria, glucosuria*)
**Glucosuria**: specifically means presence of glucose in urine when blood glucose level rises above renal threshold of *180 mg/dl*
42
Q. What is meant by qualitative & semi-quantitative benedicts test?
**Qualitative**: chemical nature of compounds present in solution
**Semi-quantitative**: reducing sugar quantity can be measure only up to 2%, not beyond this limit
43
Q. Name non-carbohydrates that give positive Benedict's test
- salicylates
- homgentisic acid
- ascorbic acid (vitamin C)
44
Q. What is the clinical application of Benedict's test? **imp**
commonly employed for both qualitative & semi-quantitative determination of reducing sugars especially **glucose in urine**.
It is a **diagnostic test for determination of glucosuria in diabetic patients**.
*The color of the
obtained precipitate gives an idea about the quantity of sugar present in the solution.*
45
principle of **barfoed's test**
**qualitative test** performed to **detect reducing monosaccharides in acidic medium**.
it can **differentiate b/w monosaccharide and disaccharides by controlling the time of heating**.
**principle**: copper acetate in presence of acetic acid is converted from divalent to monovalent state (ie CuOH2 is converted to cuprous oxide) and **red precipitate forms**
*test should't be carried out with Cl- ions in the solution as they can interfere with the test*
46
interpretation of **barfoed's test**
formation of a **green, red, or yellow precipitate** is **positive test for reducing monosaccharides**
if precipitate appears **after 5 min**, indicates **presence of disaccharide**
47
Q1. how does barfoed's test differ from Benedict's test?
Barfoed's test is also copper reduction test but unlike Benedict's test, it is **carried out in an acid (pH 4.6) rather than alkaline solution**
- can also allow you to **distinguish b/w mono & disaccharides** while Benedicts cant do that
48
Q2. How does barfoed's test distinguish between mono and disaccharides?
**Monosaccharides**: react very fast and give result within 5 minutes
**Disaccharides**: need sufficient time (more than 05 minutes) to hydrolyze into their components before they
give the positive result
49
Q3. Why in Barfoed's test monosaccharides give a quick response than disaccharides?
*test is specific for monosaccharides*
Due to **weak acidic nature of Barfoed's reagent**, it is **quickly reduced by monosaccharides**.
50
Q4. What are the causes of glucosuria?
*small increases in urine glucose levels after a large meal are not always a cause for concern*
1. **Diabetes Mellitus** (diabetes full name)
2. **Pregnancy** -- Up to half of women have glucose in their urine at some time during pregnancy
- *glucose in the urine may mean that a woman has gestational diabetes*
3. **Renal Glycosuria**: rare condition in which glucose is excreted from the kidneys into the urine, even when blood glucose levels are normal
51
principle of **seliwanoff's test**
**distinguishes b/w ketoses & aldoses**
test is positive for ketone sugars, disaccharides like sucrose will give positive test after hydrolyzing into glucose & fructose
Keto hexoses on treatment with HCl (present in seliwanoffs reagent) get **dehydrated to form 5-Hydroxy methyl furfural**
while **pentoses yield furfurals**.
these furfurals **react with Resorcinol** and give a cherry red colored complex indicating the **presence of Keto sugar**
*Fructose + HCI → 5-hydroxymethyl furfural + Levulinic acid*
*5-hydroxymethyl furfural + Resorcinol → cherry red coloured complex*
52
seliwanoff's test interpretation
appearance of **cherry red color** within 30 seconds indicates **presence of fructose** (its a ketose sugar)
*avoid overheating as aldohexoses may be converted into ketohexoses giving false positive result*
53
osazone test
performed for **identification of individual reducing monosaccharide or disaccharide** having potentially active Aldehyde or keto group in given solution by the **formation of osazone crystals**
all reducing carbohydrates react in an acidic medium at boiling temperature (100°C) with Phenylhydrazine to form their respective hydrazones which crystallize into **yellow color distinctive shape crystals on cooling**
*color comes out the same for all (yellow) but the crystals are what look different under a microscope*
54
identification of osazone crystals (shapes + time taken)
**imp also for slide identification**
*differentiated by the shape of crystals observed under microscope*
**Glucosazone crystals**: long, thin, needle shaped crisscrossing each other
- *glucose, fructose, mannose*
- *time for crystallization: 10-15 mins*
**Galactosazone crystals**: also needle shape but appear to radiate from thin center (needles look a bit more box like to me)
- *galatose*
- *time: 20 mins*
**Lactosazone crystals**: described as hedgehog shape or having a puffball appearance or pincushion with pins
- *maltose*
- *time: 15 mins after cooling*
**Maltosazone crystals**: petal like and have a flower like pattern and they are very small as compared to others, formed with difficulty
- *lactose*
- *20-25 mins after cooling*
55
Q. Why do glucose and fructose form identical glucosazone?
Crystals derived from glucose, fructose, and mannose are **identical osazones**, called
"glucosazone"
this is because except "C1" & "C2" of aldehyde & keto groups that take part in the reaction, the rest of structure in regard to "C3" to "C6" is the same for all these carbohydrates, and **this difference is removed in glucosazone crystal formation by Phenyl hydrazine**
56
Q. What is galactosemia & give its biochemical features?
inborn metabolic disorder due to **deficiency of enzyme
"uridyl
transferase"** = **accumulation of "galactose-I-phosphate"** in liver, brain tissue and lens = **cirrhosis of liver, mental retardation, and cataract**
- autosomal recessive disorder that can be diagnosed by this test
57
Q. what is lactose intolerance & give its biochemical features? + lactosuria
metabolic disorder due to **deficiency of enzyme "lactase"** = **accumulation of lactose in gut** = abdominal pain, distension, diarrhea, lactosuria.
**Lactosuria**: presence of lactose in urine
- may be seen in lactating mothers & during 3 trimester of pregnancy physiologically due to its raised levels in blood
58
Q. clinical significance of **osazone test**
significant for **diagnosis of galactosemia & lactose intolerance** in the given sample (urine) by osazone crystal formation
59
Q. name some properties exhibited by all reducing carbohydrates
- reduction
- osazone formation
- mutarotation
60
Q. what is fructosuria?
**presence of fructose in urine**
- occurs in **Fructose intolerance due to deficiency of enzyme Aldolase B & Fructokinase**.
*Urine gives Benedict's test & seliwanoffs test positive*
61
Principle of **biuret's test**
**specific for peptide bonds** - dipeptides do not give positive test!!
used to **detect the presence of proteins and peptides** (i.e. peptide bonds) by treating them with an **alkaline solution of dilute copper sulfate**.
**formation of a purplish violet colored complex of
proteins with CuSO, in a strongly alkaline solution**
- name of test derived from a specific compound **biuret**: simplest compound formed by two urea molecules and gives this
test positive
62
interpretation of **biuret's test**
**appearance of violet/purplish color indicates presence of proteins**
blue color = **test negative** & no proteins are present
**peptones** (water soluble products derived from partial hydrolysis of proteins) require **addition of large amount of copper sulfate** to give positive result (*fewer bonds so won't react as fast = need to add more*)
63
principle of **ninhydrin test**
test is often used to **detect alpha-amino acids** (*same C atom has both the amino & COOH group*)
*ninhydrin is a strong oxidizing agent so have to be careful*
Ninhydrin (triketohydrindene hydrate) **oxidizes amino acids into aldehydes** (pH range 4-8), **ammonia and CO2** through a series of reactions
**Ninhydrin itself changes to a partially reduced
from** ie hydrindantin
Ninhydrin then reacts with ammonia & hyarindantin to produce an
**intensely blue or purple pigment**, sometimes called **ruhemann's purple**.
Proline and hydroxyproline (imino acids) give yellow color (**exception - doesnt detect these**)
64
Q. why do proline & hydroxypoline give a yellow color in ninhydrin test?
because they do not have an alpha amino group
65
interpretation of **ninhydrin test**
cloudy purple color appears which indicates presence of amino acids
66
Q. Which compound is responsible for blue color in positive Ninhydrin test?
Rheumann's purple is formed by condensation of **2 molecules of ninhydrin with 1 molecule of ammonia from amino acid** & is responsible for the violet color
67
principle of **xanthoproteic test**
used to **detect aromatic amino acids (tryptophan & tyrosine)**
**Concentrated nitric acid** reacts with the **aromatic rings** that are derivatives of benzene giving the **nitration reaction** characteristic.
Tyrosine and tryptophan contain activated benzene rings which
are easily nitrated to **yellow colored compounds**
Whereas **phenylalanine does not react** with nitric acid as it contains **inactive benzene ring** therefore it will not react
68
interpretation of **xanthoproetic test**
**orange ring at the top** of liquid (yellow at the bottom) shows positive test
69
Q. what is meant by activated benzene ring?
Since benzene is an aromatic compound, it is **highly stable which means it has to be activated**.
Benzene is activated by **adding electron donor groups** to the ring (like -OH, -CH3).
The electron donor groups help to maintain resonance of the ring by delocalizing electrons into it while the reaction occurs.
70
Q. Why phenylalanine give negative test?
Its ring has substituent deactivating groups, which **deactivate the ring by the inductive effect** in the presence of an electronegative atom that withdraws the electrons away from the ring: the **activation energy increases and slows down the reaction**
71
**millon nasse's test** principle
test is **specific for tyrosine** (only amino acid containing a phenol group)
Compounds containing **hydroxybenzene radical react with reagents to form red complexes**.
Thus, this test is specific for the amino acid tyrosine and the protein containing this amino acid.
**Tyrosine** when reacted with acidified mercuric sulphate solution **gives yellow precipitate of mercury-amino acid complex**.
**addition of sodium nitrite solution and heating** = yellow complex of mercury-amino acid complex converts to mercury phenolate which is in **red color**
72
interpretation of **millon nasse's test**
brick red solution indicates presence of tyrosine
73
Q. would all phenols give positive millon's test result?
Yes, all phenols (compound having benzene ring & OH attached to it) give positive results
74
**lead sulphide test** principle
test is **specific for -SH (thiol) containing amino acids** (cysteine & cystine)
Some of sulfur in cystine is converted to sodium sulfide (Na2S) by boiling with 40% NaOH.
The Na2S can be detected by the precipitation of PbS from an alkaline solution.
When the amino acids containing sulfhydryl group are heated with base, the sulfhydryl group and disulfhydryl are directly converted to inorganic sulfur.
Which is **confirmed by the black precipitate** of PbS (lead sulfide) when adding lead acetate Pb(CH,COO)2.
75
**lead sulphide test** interpretation
formation of black precipitate will indicate presence of -SH (thiol) containing amino acids
76
Q. which amino acids contain -SH group?
cysteine & methionine
77
Q. what is the difference b/w cysteine and cystine?
**Cysteine**: alpha-amino acid.
When 2 Cysteine amino acid residues form a disulfide bond, the **resulting dimeric residue is known as cystine**.
*Cystine is responsible for the tertiary structure
of proteins*
78
Q. Homocystinuria is a defect of which amino acid?
Methionine
79
Q. What is cystinuria?
Inability to reabsorb **cystine, ornithine, arginine, lysine** from proximal renal tubule and their **excretion in urine**
80
What is chromatography?
name given to a set of techniques by which **members of a group of similar substances are separated by continuous redistribution** between two phases (**stationary and mobile phase**)
- separation depends upon relative tendencies of molecule in a mixture to associate more strongly with one plane or another phase (polarity)
81
Enumerate different types of chromatography
1. **Paper chromatography**: simple technique for amino acids detection
2. **Adsorption chromatography**: based on affinity b/w substances
- *used for separation of layer molecules such as enzymes, proteins, glycoproteins, & nucleic acids*
3. Ion-exchange chromatography
4. Partition chromatography
5. Exclusion chromatography
6. High Pressure Liquid Chromatography (HPLC)
82
stationary phase, mobile phase, capillary action in chromatography **imp**
**Stationary Phase**: the paper in paper chromatography.
- doesn't move—hence "stationary."
It's usually made of cellulose, which is a type of absorbent material.
**Mobile Phase**: the solvent (like water or alcohol).
It moves up the paper through a process called capillary action.
The mobile phase *carries the components of the mixture with it*
**capillary action**: how liquids can travel up narrow spaces, without needing an external force (like gravity).
- In chromatography, **capillary action is what pulls the solvent up the paper**, carrying different parts of the mixture with it.
83
define RP-HPLC
type of liquid chromatography used to separate, identify, and analyze compounds in a mixture—especially useful for proteins, amino acids, and drugs
In this, **the stationary phase is non-polar** (hydrophobic) and
the **mobile phase is polar**
84
Rf value in chromatography **imp** (significance + formula)
Rf: retention factor
**Rf = distance travelled by substance/ distance travelled by mobile phase**
**significance**: Rf value of unknown amino acids is compared with known amino acids
- used to identify the amino acid
85
Clinical applications of chromatography **imp** + amniocentesis
**Amino acid disorders**
**Phenylketonuria**: defect of enzyme *phenylalanine hydroxylase*
**Maple Syrup Urine Disease (MSUD)**: deficient carboxylation of branch chain amino acids
**amniocentesis**: prenatal diagnosis of these disorders is possible!!
- the concerned enzymes are detectable in a culture of amniotic fluid cells
86
how to convert mg/dl to mmol/L?
**imp- make sure to know units too**
**(mg per 100 ml) x 10 / (molecular weight) = mmol/L**
ex. convert 8.5 mg/dl to mmol/L
- molecular weight = 40
*(8.5 x 10)/(40) = 2.1 mmol/L*
*molecular weight and valency will always be given*
87
how to convert mg/100 ml to mEq/L? **imp**
**(mg per 100 ml) x 10 x valency/ (molecular weight) = mEq/Litre**
*valency & molecular weight are always given*
88
valency equations
**equivalent weight = molecular weight/valency**
*N = equivalent weight*
so can also rewrite as **M = N x Valency**
- *if valency of substance = 1 then its mEq/L will be equal to mmol/L
- if valency is higher than 1, then multiply its mEq/L by the valency number*
