laboratory exercises in medical chemistry...

Laboratory Exercises in Medical Chemistry I

1

st year, General Medicine

Faculty of Medicine in Pilsen

Charles University

Name: Confirmation of the attendance at the labs

(stamp, tutor's signature)

Study group:

Date:

Date of report delivery:

Report accepted returned for revision

Programme

a) Reactions of inorganic compounds

- reaction of Ag+ ions with dilute solution of HCl

- reaction of Fe3+

ions with a solution of potassium ferrocyanide

- reaction of Fe3+

ions with SCN- ions

- reaction of Cu2+

ions with ammonia

- reaction of Ca2+

ions with oxalic acid

- reaction of carbonates with dilute solution of HCl

b) Qualitative analysis of cations and anions

- demonstration of the procedure for identification of Ni2+

cation

- demonstration of the procedure for identification of SO42-

anion

- identification of the cation and the anion in an unknown sample

There is a rack with clean test tubes on your working place. In addition, there is an empty rack

intended to be used for placing dirty test tubes. After the work is done, take the rack with test tubes

you have used, and pour out the content according to instruction of laboratory assistants (there are

extra bottles for collecting the waste in the laboratory). Moreover, they will specify, where to put

empty dirty test tubes.

A lab coat is required to be worn over your clothes, when working in a lab!

After the work is done, clean your working place so that it is the same as it was in the beginning!

Before you leave the lab, ask laboratory assistant for a stamp and your tutor for signature!

1

a) Reactions of inorganic compounds

For each of the following exercises, you need ONE clean test tube. It does not matter on the exact measurement

of volumes. You can pour the solutions into test tubes directly from bottles in appropriate small volume

(about 1 ml, i.e. approx. 1 cm of the column height of liquid in a test tube). Do not pipette the solutions!

1. Reaction of Ag+ ions with diluted solution of HCl

Pour into a test tube about 1 ml of solution with Ag+ ions.

CAUTION: contamination of the skin with Ag+ ions causes unremovable black spots

( ! not only pouring the solution on your skin is risky, touching on dirty glass as well ! )

Add about 1 ml of dilute solution of HCl into the test tube and watch the reaction.

Describe the changes observed:

Chemical equation in ionic form Colour of the precipitate

2. Reaction of Fe3+

ions with a solution of potassium ferrocyanide

Pour into a test tube about 1 ml of solution with Fe3+

ions.

Add several drops of potassium ferrocyanide solution into the test tube and watch the reaction.


Chemical equation in ionic form Colour of the solution

What is the traditional name for a dye produced by reaction of Fe3+

ions with ferrocyanides?

2. Reaction of Fe3+

ions with SCN- ions

Pour into a test tube about 1 ml of solution with Fe3+

ions. Add several drops of a solution with SCN-.


Potassium ferrocyanide – formula:

Colour of the solution containing Fe3+

ions:

2

4. Reaction of Cu2+

ions with ammonia

Pour into a test tube about 1 ml of solution with Cu2+

ions.

Add about 1 ml of dilute solution of ammonia into the test tube and watch the reaction.


Chemical equation in ionic form Colour of the solution

5. Reaction of Ca2+

ions with oxalic acid

Free Ca2+

ions play many roles in body fluids. Ca2+

ions are very important in the process of blood

clotting (hemocoagulation). Removal of Ca2+

can prevent blood clotting in vitro. This can be used in

clinical medicine. Ca2+

ions can be removed from the solution (bound) by the use of organic acids

with more carboxylic groups, e.g. oxalic acid or citric acid.

oxalic acid – structural formula

citric acid – structural formula

Let's try the reaction of Ca2+

ions with oxalic acid. Pour into a test tube about 1 ml of Ca2+

solution.

Add about 1 ml of oxalic acid solution into the test tube and watch the reaction.



6. Reaction of carbonates with diluted solution of HCl

Pour into a test tube about 1 ml of sodium carbonate solution. Add about 1 ml of dilute solution of

HCl into the test tube and watch the reaction.


Chemical equation Gas, that is released

Colour of the solution containing Cu2+

ions:

3

b) Qualitative analysis of cations and anions

All the reactions you will perform will be in aqueous environment, where are the soluble inorganic

salts usually dissociated into ions.

In your practical exercises, you will work only with solutions, where is single "simple" inorganic

compound disolved, in other words – just one cation and just one anion. This information very

simplifies the analysis. When you successfully identify one cation, you can stop. It is not necessary tio

continue, no other cation is in the sample. Similarly for the anion. There can be often many cations

and many aniont in real samples. In such cases (mixture of several compounds), the analysis is more

difficult.

Classification of cations – hydrogene sulfide system

Cations are classified into 5 groups according to the results of so called "group reactions".You have to

use group reagents for that purpose. It is necessary to work systematically – to begin from the very

beginning – to test the presence of group I cations.

In case of positive result you know that one of the cations of this analytical group is in the sample.

Now it is time to use so called selective or specific reactions to distinguish among cations belonging

into this group.

Negative result excludes this group of cations. Only after exclusion of the group, you may continue

and test the presence of cations from the next group in order.

Not understanding the principle, on which the whole system of classification is based, is the most

usual cause for being unsuccessful in analysis of unknown sample. You can test the group reaction for

"higher" analytical group only after you exclude the presence of cations of all previous groups! Cation

of the group I can positively react with group reagents for "higher" groups!

You have to keep the order of reactions tested on your sample, i.e. you have to begin always with

tests for group I and in case of negative result to continue to the next group in order. Once the group

reaction is positive, identification of the group is done. Now you have to direct your attention only on

cations belonging into this group.

Group I – insoluble chlorides Ag+ Pb

2+

Group reagent: dilute solution of HCl

Add a dilute solution of HCl to the sample. Formation of precipitate means the presence of cation

from this group. If no precipitate forms, you can exclude this group and you have to continue with test

for presence of cations of the group II.

AgCl white precipitate

PbCl2 white precipitate

Both cations, Ag+ and Pb

2+, give white precipitate of insoluble chloride. How can we distinguish

between them?

1st option

Ag+ and Pb

2+ cations form precipitates of chromates that very differ in colour.

Ag2CrO4 reddish-brown precipitate

PbCrO4 yellow precipitate

The procedure: Pour the original solution in a new test tube and add appropriate volume of K2CrO4.

aqueous solution of sodium chloride – what ions are present?

4

2nd

option

Silver chloride is soluble in ammonia, lead chloride is insoluble.

The procedure: Add enough of dilute ammonia solution to the precipitate in the test tube (about

double volume than the volume you already have in the test tube). Mix thoroughly. If the precipitate

disappears, it was AgCl, and we have identified Ag+ cation. In the opposite case, the cation is Pb

2+.

Group II – insoluble sulfides that precipitate from acidic environment

This group has two subgroups: II. A Hg2+

Cu2+

Cd2+

II. B As3+

Sb3+

Sn2+

Group reagent: dilute solution of HCl and hydrogensulfide water

Acidify the sample by addition of small volume of dilute HCl solution. Go to the fume chamber and

add hydrogen sulfide water. Formation of precipitate of insoluble sulfide means presence of a cation

of this group.

group II.A HgS black precipitate

CuS black precipitate

CdS yellow precipitate

group II.B As2S3 yellow precipitate

Sb2S3 orange precipitate

SnS brown precipitate

Distinguishing between subroups

Precipitates of sulfides of II.A group cations are insoluble in ammonium polysulfide (NH4)2Sx.

Precipitates of sulfides of II.B group cations are soluble in ammonium polysulfide (NH4)2Sx.

There is a bottle with (NH4)2Sx in a fume chamber. Be very careful in testing the solubility in case of

yellow precipitate (the only way to distinguish between Cd2+

and As3+

).

Do NOT take the test tube out from the fume chamber! When the experiment is done, pour the content of the test tube into a bottle for collection of waste

inside the fume chamber and put the ampty test tube into a basket there

Ions in subgroups can be distinguished according to the colour of precipitates of sulfides. Hg2+

ions

and Cu2+

ions can be distinguished form each other according to the colour of original solution. Hg2+

ions are colourless Cu2+

ions form in aqueous solutions blue complexes [Cu(H2O)4]2+

. The blue colour

can be deepened by addition of ammonia.

Group III.A – insoluble hydroxides Fe3+

Cr3+

Al3+

Group reagent: solution of NH4Cl and dilute ammonia (NH3)

Optimal pH for precipitation is around 9, the reagent acts as a buffer and mantaines this wanted pH.

Add NH4Cl soloution to the sample in a test tube first and then add dilute solution of ammonia.

Formation of precipitate means presence of cation from this group.

Fe(OH)3 brown precipitate

Cr(OH)3 grayish-green precipitate

Al(OH)3 white (colourless) gelatinous precipitate

Use the colour of original solution to distinguish between cations of this group. Fe3+

ions are yellow in

solution, Cr3+

ions are deep green and Al3+

ions colourless.

5

Presence of Fe3+

ions can be confirmed by two nice reactions. Use original solution of the sample, add

ammonium thiocyanate (deep red colour similar to blood is formed) or potassium ferrocyanide (deep

blue colour is formed, so called "Prussian blue").

[Fe(SCN)]2+

red solution

{Fe[Fe(CN)6]}- blue solution

Group III.B – sulfides that precipitate in alkaline pH Fe2+

Co2+

Ni2+

Zn2+

Group reagent: dilute ammonia and solution of (NH4)2S

Ions of group III.B precipitates as sulfides insoluble in alkaline environment. First add small volume

of ammonia and then the precipitation reagent – amonnium sulfide (work inside fume chamber).

Formation of precipitate means presence of cation from this group.

FeS black precipitate

CoS black precipitate

NiS black precipitate

ZnS white precipitate

Do NOT take the test tube out from the fume chamber! When the experiment is done, pour the content of the test tube into a bottle for collection of waste

inside the fume chamber and put the ampty test tube into a basket there.

You should easily identify Zn2+

ions, that gives whitesulfide and are colourless in original solution

(all the other cations in this group are obviously coloured). The appeearance of other three sulfides is

similar (black precipitate), so we have to continue in investigation.

Fe2+

ions are light green in colour and give striking colour raction with potassium ferricynide (deep

blue colour - "Prussian blue").

{Fe[Fe(CN)6]}- blue solution

Co2+

ions are pink in hydrated form.

Ni2+

ions are easy to identify, their aqueous solutions are clearly green. Addition of few drops of

ammonia changes the colour to blue.

[Ni(NH3)6]2+

blue solution

Group IV – insoluble carbonates Ba2+

Sr2+

Ca2+

Mg2+

Group reagent: dilute solution of ammonia and a solution of (NH4)2CO3

First add small volume of ammonia and then the precipitation reagent – amonnium carbonate

(NH4)2CO3. Formation of precipitate means presence of cation from this group.

BaCO3 white precipitate

SrCO3 white precipitate

CaCO3 white precipitate

MgCO3 white precipitate (after being boiled)

Ca2+

precipitates after addition of oxalic acid. The other cations do not react.

(COO)2Ca white precipitate

To distinguish between Ba2+

and Sr2+

use the flame test. There is a working place with lab burner in

the laboraotry. Ba2+

ions turn colour of the flame to green, Sr2+

to crimson (red).

6

Group V - ions of alkali metals and ammonium ion Li+ Na

+ K

+ NH4

+

Group reagent: – (no group reagent)

Cation is classified to be from fifth group of cations by excluding all the previous groups. It is quite

difficult to identify cations of group V.

Distinguishing among Li+, Na

+ and K

+ can be done by the flame test. Li

+ ions colour the flame to

crimson (red). (caution – very similar colour is caused by Sr2+

). Na+ ions colour the flame to yellow

and K+ ion to violet.

NH4+ ions can be detected by Nessler's reagent (alkaline solution of Hg[HgI4]). This reaction is very

sensitive, even very diluted solutions react. Pour about 1 ml of unknown sample into a test tube, add

small volume of dilute solution of NaOH and few drops of the reagent. Record the formation of brown

precipitate, in case of dilute solutions of yellow colour.

Classification of cations into analytical groups

Group Reagent Cation Colour of the precipitate Original solution Notes

I HCl Ag

+ AgCl (white) colourless reddish-brown Ag2CrO4

Pb2+ PbCl2 (white) colourless yellow PbCrO4

II.A

HCl

+

H2S

Hg2+ HgS (black) colourless

precipitates of sulfides are

insoluble in ammonium

polysulfide

Cu2+ CuS (black) light blue

Cd2+ CdS (yellow) colourless

II.B

As3+ As2S3 (yellow) colourless

precipitates of sulfides are

soluble in ammonium

polysulfide

Sb3+ Sb2S3 (orange) colourless

Sn2+ SnS (brown) colourless

III.A

NH4Cl

+

NH3

Fe3+ Fe(OH)3 (brown) yellow reaction with [Fe(CN)6]

4-, SCN

-

Cr3+ Cr(OH)3 (grayish-green) green

Al3+ Al(OH)3 (colourless gelatinous) colourless

III.B

NH3

+

(NH4)2S

Fe2+ FeS (black) greenish-yellow reaction with [Fe(CN)6]

3-

Co2+ CoS (black) pink

Ni2+ NiS (black) green

Zn2+ ZnS (white) colourless

IV

NH3

+

(NH4)2CO3

Ba2+ BaCO3 (white) colourless flame test: green

Sr2+ SrCO3 (white) colourless flame test: crimson (red)

Ca2+ CaCO3 (white) colourless reaction with oxalic acid

Mg2+ MgCO3 (white) after boiling colourless

V –

Li+ – colourless flame test: crimson (red)

Na+ – colourless flame test: yellow

K+ – colourless flame test: violet

NH4+ – colourless Nessler's reagent

7

Classification of anions

To succeed in identification of anion, you have to work systematically. Classification into analytical

groups is based on results of precipitation reactions with two reagents: AgNO3 and Ba(NO3)2. The

strategy is very different from the one used for cations. In case of anion, it is simpler:

Take two test tubes. Into both of them, pour about 1 ml unknown sample. Add about 1 ml of AgNO3

solution in one of them, add about 1 ml of Ba(NO3)2 solution in the other one. You can directly

identify the analytical group according to the results of these two reactions (only for groups IV, V.A

and V.B you have to do one more step – to test the solubility of the precipitate formed).

Group I SO42-

reaction with: Ag+ no reaction

Ba2+

white precipitate

You can confirm the result by the reaction with Pb2+

ions. White precipitate of PbSO4 is formed.

Group II PO43-

CrO42-

OH-

reaction with: Ag+ coloured precipitate

Ba2+

white or yellow precipitate

These anions give deeply coloured, water insoluble silver salts - yellow Ag3PO4, reddish-brown

Ag2CrO4 and dark brown AgOH that immediately decomposes to Ag2O. Precipitates of barium salts

are white or yellow.

Group III SO32-

CO32-

reaction with: Ag+ white precipitate

Ba2+

white precipitate

In this group, white precipitates are formed with both reagents, only the precipitate of Ag2CO3 is dirty

yellow. Precipitates of silver salts are easily soluble in both dilute HNO3 and ammonia.

The result of group reactions is not reliable for distinguishing between these two anions. It is

necessary to conduct one more test. Sulfites have reducing properties, carbonates not. Use iodine

solution for detection of reducing properties. Sulfites reduce the brown iodine solution into colourless

iodide ions. Carbonates do not react.

Procedure: Pour into a test tube about 1 ml of sample to be tested, add dropwise iodine solution (use

the dropper) and watch the reaction.

Group IV NO2- S

2-

reaction with: Ag+ white or black precipitate

Ba2+

no reaction

Sulfides can be easily recognized because of black, very insoluble precipitate of Ag2S. You have to be

more careful in case of nitites. Very similar white precipitates are are formed in case of many other

anions of the groups V. A and V. B. To distinguish among them, note that precipitate of AgNO2 is

soluble in dilute solution of nitric acid (HNO3).

Group V.A Cl- [Fe(CN)6]

3-

reaction with: Ag+ white or brown precipitate (insoluble in HNO3, soluble in ammonia)

Ba2+

no reaction

You should easily identify ferricyanides. Original solution is light yellow, precipitate of silver salt is

brown. Moreover, for definite confirmation, you can use the reaction with Fe2+

ions (formation of

"Prussian blue"). White precipitate of AgCl must be tested for solubility, to distinguish from

precipitates formed from NO2- and anions of group V.B. AgCl is insoluble in dilute HNO3, but soluble

in ammonia.

8

Group V.B Br- I

- SCN

- [Fe(CN)6]

4-

reaction with: Ag+ white or yellow precipitate (insoluble in HNO3, insoluble in ammonia)

Ba2+

no reaction

You can record light yellow colour of AgI. Ferrocyanides ([Fe(CN)6]4-

) are light yellow in solution.

Distinguishing among anions of this group

The easiest way how to distinguish among these anions, is to use solution of FeCl3. Bromides do not

react, iodides are oxidized to iodine (solution in a test tube turns brown), thiocyanates give deep red

solution of [Fe(SCN)]2+

and ferrocyanides form deep blue colour ("Prussian blue").

Group VI NO3- MnO4

-

reaction with: Ag+ no reaction

Ba2+

no reaction

Distinguishing between these two anions is easy accordind to the colour of original solution. Nitrates

(NO3-) are colourless, permanganates (MnO4

-) are deep violet, pink when diluted.

Classification of anions into analytical groups

Group Anion

Colour and solubility of precipitate formed with

Notes

AgNO3 Ba(NO3)2

I SO42-

– white with Pb2+

- white precipitate PbSO4

II

PO43- yellow white

CrO42- reddish-brown yellow with Pb

2+ - yellow precipitate PbCrO4

OH- brown white

III SO3

2- white white reduces brown I2 to colourless I-

CO32- white white

IV NO2

- white soluble

in HNO3

–

S2- black –

V.A Cl

- white insoluble

in HNO3

soluble in NH3

–

[Fe(CN)6]3- brown – with Fe

2+ --> "Prussian blue"

V.B

Br- white

insoluble

in HNO3

insoluble

in NH3

– with Fe3+

- no reaction

I-

light

yellow – with Fe

3+ --> formation of brown I2

SCN- white – with Fe

3+ --> red colour

[Fe(CN)6]4-

white – with Fe3+

-->"Prussian blue"

VI NO3

- – – original solution colourless

MnO4- – – original solution deep violet

It is difficult to distinguish among NO2-, Cl

- a Br

-. These three anions are precipitated only as

insoluble silver salts. In all three cases, the result is white precipitate. To distinguish among them, it is

necessary to divide the precipitate of silver salt into two test tubes and check the solubility of it in

dilute nitric acid (HNO3) and in dilute ammonia (NH3) – see the table.

9

Where to find the solutions needed?

Working place: group reagents for cation classification: HCl

NH4Cl

(NH4)2CO3

group reagents for anion classification: AgNO3

Ba(NO3)2

for solubility tests of precipitates: dilute solution of ammonia (NH3)

dilute solution of nitric acid (HNO3)

solution of iron (III) chloride (FeCl3)

Fume chamber: hydrogensulfide water H2S

ammonium polysulfide (NH4)2Sx

ammonium sulfide (NH4)2S

Place with shared solutions: potassium chromate (K2CrO4)

ammonium thiocyanate (NH4SCN)

potassium ferrocyanide (K4[Fe(CN)6])

potassium ferrocyanide (K3[Fe(CN)6])

oxalic acid

Nessler's reagent

solution of Pb2+

solution of iodine

Do NOT take anything away from the place with reagents! What is there is shared with others!

The correct way: Come with your test tube. Add what you need at this place.

Do NOT take anything out from the fume chamber!

The correct way: Come with your test tube to the fume chamber. Conduct an experiment. Remember

the result. Pour the content of the test tube into the bottle for waste and place empty test tube into a

basket located inside the fume chamber.

A. Demonstration of the procedure for identification of Ni2+

cation and SO42-

anion

The following part of the manual shows the procedure of identification on "known samples" What

will be demonstrated, are the steps resulting in identification of Ni2+

cation and SO42-

anion.

There is a sample marked Ni2+

and a sample marked SO42-

on your working place. Now you have the

opportunity to try all the procedure on these samples.

Demonstration of the procedure for identification of Ni2+

In the procedure of cation identification, it is necessary to keep the order of group reactions tested

(i.e. begin with group I and continue towards "higher" groups, until the reaction is positive). After

identification of the group, you do NOT continue in testing of reactions for "higher" groups! Now, the

only step missing to fulfill the final goal, is to distinguish among cations belonging into the identified

group. As an unknown sample, you will use "Ni2+

".

10

1. Reagent for group I cations: dilute solution of HCl

Pour into a test tube about 1 ml of unknown sample solution (Ni2+

in this case). Add small volume of

dilute solution of HCl.


Positive reaction would be manifested by formation of precipitates of insoluble chlorides. Negative

result excludes presence of cations of this group. We have to continue and test the group reaction for

group II of cations.

2. Reagent for group II cations: dilute solution of HCl and hydrogensulfide water



dilute solution of HCl. Go to the fume chamber with this test tube and add hydrogensulfide water.


Positive reaction would be manifested by formation of coloured precipitates of insoluble sulfides. For

cations of group II.A, precipitates would be insoluble in ammonium polysulfide (NH4)2Sx. For cations

of group II.B, precipitates would be soluble in ammonium polysulfide. Negative result excludes

presence of cations of this group. We have to continue and test the group reaction for group III.A of

cations.

3. Reagent for group III.A cations: solution of NH4Cl and dilute ammonia solution



NH4Cl solution. For the precipitation, optimal pH is around 9. Ammonia itself would be more

alkaline, we need to add NH4Cl first. Then you can add about 1 ml of dilute ammonia solution.


Positive reaction would be manifested by formation of precipitates of insoluble hydroxides. Negative

result excludes presence of cations of this group. We have to continue and test the group reaction for

group III.B of cations.

4. Reagent for group III.B cations: solution of (NH4)2S and dilute ammonia solution



dilute ammonia solution.


Go to the fume chamber with this test tube and add ammonium sulfide. Record positive reaction –

formation of coloured precipitate of the sulfide.


Conclusion: Our cation belongs to the group III.B. What it could be: Fe2+

, Co2+

, Ni2+

or Zn2+

.

11

One of these can be excluded because of the colour of sulfide precipitate. Which one?

What is helpful now? Colour of analysed sample.

Colour:

According to the colour of the original sample solution, you can easily identify Ni2+

cations. For

confirmation, you can test the reaction of the sample with ammonia. Pour into a test tube about 1 ml

of original unknown sample solution (Ni2+

in this case). Add dilute solution of ammonia (NH3).


Chemical equation in ionic form

Demonstration of the procedure for identification of SO42-

As an unknown sample, you will use "SO42-

". Similarly to cations, also anions can be classified into

several groups on the basis of the results of the reactions with group reagents. For anions it is

relatively easy, there are only two: AgNO3 and Ba(NO3)2.

Prepare two test tubes. Into both of them, pour about 1 ml unknown sample (SO42-

in this case).

Colour of the sample:

1) reaction with Ag+

Take one of the test tubes prepared and add about 1 ml of AgNO3 solution.


2) reaction with Ba2+

Take the second test tube prepared and add about 1 ml of Ba(NO3)2 solution.



It is very easy in this case. Combination of the results of reactions with Ag+ and Ba

2+ clearly points to

the Group I of anions. There is a single anion: SO42-

.

12

B. Analysis of unknown sample

There is a test tube marked with number of your sample in a rack on the working place. The test tube contains unknown

sample to be identified. First, try to identify the cation, and the anion after that. Be responsible, volume of the sample you

have is limited. And it must be enough to perform all the tests needed for definite identification. Do not put anything into

the original test tubes with sample. For individual tests, put a small volume of the sample into new clean test tube!

No new sample available at all!

It may happen that either cation or anion (or both) cause the sample to be coloured.

Sample number: Colour of the sample:

CAUTION: Colour of the solution caused by presence of coloured cations may influence appearance of precipitates

formed in identification of anions and vice versa, e.g. tested reaction for anion identification should give white precipitate,

but this precipitate may look "bluish" because of the presence of blue cation! This should be taken into account!

Describe the systematic procedure leading to definite identification of cation present in the unknown sample.

(all the test performed, including results; chemical equations in ionic form)

Cation Formula Name in words Analytical group

Describe the systematic procedure leading to definite identification of anion present in the unknown sample.

(all the test performed, including results; chemical equations in ionic form)

Anion Formula Name in words Analytical group

CONCLUSION

Analysed unknown sample is a solution of: (give the inorganic compound)

Formula

Name in words

laboratory exercises in medical chemistry...

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