PREPARATION OF CHROMATOGRAPHY SPRAY

REAGENTS
CHROMATOGRAPHY SPRAY INDEX
SELECTION GUIDE
Acids organic

Bromocresol Green

Aldehydes

Fuchsin

Alkaloids

Dragendorff's; Iodoplatinate

Amines

Iodoplatinate; Ehrich's; Ninhydrin

Amino acids and related

Ninhydrin
compounds
Antioxidants

Phosphomolybdic acid

Barbiturates

Mercuric diphenylcarbazone

Flavanoids

Antimony trichloride

Glycopids

Bial's; Orcinol; Diphenylamine

Lipids

Antimony trichloride; Bial's; Orcinol; Bromothymol blue; Cupric

acetate; 2,7-Dichlorofluorescein; Diphenylamine; Dragendorff's;
Molybdenum blue; Ninhydrin; Potassium- Dichromate/H2SO4;
Rhodamine B; Phodamine 6G

Phenols

Phosphormolybdic acid; Rhodamine B

Steroids

Antimony pentachloride; Antimony trichloride; Phosphormolybdic

acid

Terpenes

Antimony pentachloride

Vitamins

Antimony pentachloride; Iodoplatinate

Charring Reagent

Potassium dichromate/H2SO4

ALPHABETICAL SEARCH FOR TLC VISUALIZATION SPRAY REAGENTS

Aluminium chloride
For flavonoids
Dissolve 1 gm aluminum chloride in 100 ml 95% ethanol
Look for yellow fluorescence in UV light (360nm)
4-Aminoantipyrine/potassium hexacyanferrate (III) (Emerson reaction) for the detection of phenols
and aryl amines
Solution I: 1g aminoantipyrine (4-aminophenazone) in 100ml 95% ethanol
Solution II: 4g potassium hexacyanoferrate (III) in 50 ml water and 50ml 95% ethanol
Spray with solution I
Dry 5 minutes with warm air(heat gun)
Spray with solutionn II
Dry again for 5 minutes
Place plate in an ammonia vapor chamber (25% ammonia solution) to develop.
Red-orange to pink spots develop.
2-Aminoethyl diphenylborate, see Ethanolamine diphenylborate
Ammonia
25% w/v in water.For neutralizing ninhydrin fixer for chromatograms and numerous other applications.
Ammonium metavanadate, or ammonium monovanadate (see Vanadium(V) / sulfuric acid)
Ammonium molydbate For detection of phosphoric acid derivatives
Solution I: 10 mls perchloric acid in 50 ml water and 50 ml acetone
Solution II: ammonium molybdate soln: 5g (NH4)6Mo7O24.4 H2O in 40 ml 1:1 nitric acid and 60ml water.
Solution III: (Stannous chloride)Tin (II) chloride soln: Dissolve 0.5g SnCl 2.2 H2O in minimum conc. HCl
and make up to 100 ml solution.
Dry developed chromatogram and heat to 60 C in TLC oven.
Spray twice with perchloric acid reagent.
Then spray the still warm plate with ammonium molybdate solution (solution II) followed by the Tin II
chloride reagent. (solution III)
Phosphates appear as blue-green spots. Polyphosphates can be detected by using molybdenum blue
spray reagent.
Aniline-Diphenylamine For detecting reducing sugars.

Dissolve 1.8gm aniline and 1.8gm diphenylamine in 80ml acetone and add 20ml conc.HCl.
Aniline phthalate For the detection of reducing sugars
Dry the chromatogram
Spray with 0.93g aniline and 1.66g o-phthalic acid dissolved in 100ml n-butanol saturated with water.
Briefly dry with hot air, then heat to 110C for 10 minutes
Spots show different colors. Some spots give fluorescence at 365nm.
p-Anisaldehyde / sulfuric acid For detection of phenols, sugars, steroids, and terpenes
Spray with a solution of freshly prepared 0.5ml p-anisaldehyde in 50ml glacial acetic acid and 1ml conc.
sulfuric acid.
Heat to 105C until maximum visualization of spots. Enhance background with water vapor spray.
Components give violet, blue, red, grey or green spots.
p-Anisaldehyde / ethanol For detection of sugars
Spray with a solution of freshly prepared 1ml p-anisaldehyde, 1ml conc. sulfuric acid in 20ml ethanol.
Heat at 110C.
Sugar phenylhydrazones give green-yellow spots in less tham 5 mins. Sugars will produce blue, green,
violet spots in 10min.
p-Anisidine Hydrochloride For detection of carbohydrates / sugars
Mix a solution of 3gm p-anisidine hydrochloride in 100ml n-butanol
Spray and heat at 100C.
Aldohexoses are seen as green-brown spots, ketohexoses as yellow spots, aldopentoses as green
spots, and uronic acids as red spots.
Anisidine phthalate For detection of carbohydrates and reducing sugars
Spray with a solution of 1.23 g p-anisidine and 1.66g phthalic acid in 100ml 95% ethanol.
Hexoses, green; pentoses, red-violet, methylpentoses, yellow-green; uronic acids,brown.
Antifoam
25% antifoam reagent in methylene chloride.
For preventing/dispersing foam.
Antimony (III) chloride For detection of vitamins A & D, carotenoids, steroids, sapogenins, steroid
glycosides, terpenes
Spray with a saturated solution of 25g antimony (III) chloride in chloroform.
Heat 10min at 100C, then view at 360nm.
B
Bial reagent See Orcinol
Boute reaction for detection of phenols
Reaction of constituents with nitrogen dioxide and ammonia vapor
Dry and heat the developed chromatogram
Place hot plate for 3 10 minutes in a chamber with NO2 vapour (from conc. nitric acid)
Then treat with NH3 vapour (from conc. ammonia)
Bromine / Carbon tetrachloride For detection of organothiophosphorous pesticides
Place chromatogram in a chamber with a 10ml bromine in 90ml tetrachloride. No contact with the liquid.
Bromocresol Green
Dissolve 0.04gm bromocresol green in 100ml ethanol
For detecting organic acids and bases
Bromocresol green For tank development of organic acids
Dip chromatogram in a solution of 0.1g bromocresol green in 500ml ethanol and few drops dilute NaOH
Acids yield yellow spots on a blue background.
Bromthymol blue For detection of lipids and phospholipids

Dissolve 0.1gm bromthymol blue in 10% aqueous ethanol made just alkaline with NH4OH
Spray dried plate.
Compounds produce blue-green colors.
C
Chloranil reagent (tetrachloro-p-benzoquinone) For detection of phenols
Spray with a solution of 1gm tetrachloro-p-benzoquinone in 100ml toluene
Chlorine / o-tolidine For detection of chloroamine forming compounds, e.g., urea derivatives,
carbamated, antibiotics
Solution I: Dissolve 160mg o-tolidine in 30ml glacial acetic acid, add 500ml distilled water, and add 1gm
KI
Solution II: saturated solution of o-tolidine in dilute acetic acid with 0.85gm KI added.
Chloramine-T
Dissolve 5gm of reagent and 0.5gm sodium hydroxide in 100ml water.
For detection of phenolic compounds.
Cupric Acetate
Dissolve 3gm cupric acetate and 15mls phosphoric acid in 85mls water..
For detecting prostaglandins.
Copper sulfate / phosphoric acid Used as a charring reagent for polymer bound TLC plates.
Spray with a solution of 10gm copper (II) sulfate 10mls phosphoric acid in 90mls water.
Heat 5-30min at 110C
View every few minutes to see if colored or fluorescent spots at 254 and 360nm appear.
Spots become brown, grey or black.
Chromosulfuric acid See under Potassium dichromate / sulfuric acid
a-Cyclodextrin
Dissolve 1gm of the reagent in 100ml ethanol.
Distinguishes saturated from unsaturated lipids.
D
DDQ Reagent (Dichlorodicyanobenzoquinone) For detection of phenols
Spray with a solution of 2gm 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in 100ml toluene
2 Dichlorofluorescein
Dissolve 0.2gm of the reagent in 100ml ethanol.
For detecting saturated and unsaturated lipids.
Dichlorofluorescein For the detection of sweeteners saccharine and cyclamate
Dissolve 0.2gm of dichlorofluorescein in 100ml ethanol
Spray and dry with warm air.
View under 360nm UV light
Dichlorofluorescein / fluorescein sodium salt For detection of N-substituted barbiturates
Spray with a solution of 0.1gm dichlorofluorescein in 100ml ethanol
Then spray with a solution of 0.1gm fluorescein sodium salt in 100ml ethanol
2,6-Dichloroquinone -4- chloroimide For detection of antioxidants, phenols, amines, aromatic
hydrocarbons, pharmaceuticals, phenoxyacetic acid herbicides, etc
Spray with a freshly prepared solution of 1.0gm 2,6-dichloroquinone-4-chloroimide in 100ml ethanol.
Heat 10min at 110C and treat with NH3 vapor
p-Dimethylaminobenzaldehyde(Ehrlich reagent). For detection of sulfonamides, amines, indoles, and
ergot alkaloids

Dissolve 1gm p-dimethylaminobenzaldehyde in 50ml hydrochloric acid and add 50ml methanol
Heat plates for 20min at 50C
4- DimethylaminocinnamaldehydeFor detecting indoles.
Dissolve 0.2gm of the reagent in 80ml ethanol and 20ml conc. HCl.
N,N-Dimethyl-p-phenylenediamine dihydrochlorideFor detecting organic peroxides.
Dissolve 1gm of the reagent in 80ml methanol and 20ml conc. HCl.
Diphenylhexatriene For detecting lipids.
Dissolve 0.03gm of the reagent in 100ml chloroform.
2,4-Dinitrophenylhydrazine For detection of aldehydes and ketones
Spray plate with solution of 0.4 g 2,4-DNPH in 100ml 2N hydrochloric acid, add 1ml ethanol
Observe yellow-red spots.
Diphenylamine For detection of glycosides, glycolipids
Dissolve 5gm diphenylamine in 50ml ethanol, add 40ml conc. HCl and 10ml glacial acetic acid
Spray and cover plate with another glass plate, heat 30-40min at 110C until spots appear
Glycolipids produce blue spots.
s-Diphenylcarbazone For detection of barbiturates
Spary with a solution of 0.1gm s-diphenylcarbazone in ethanol
Barbiturates give purple spots
Diphenylcarbozone/Mercuric sulphate For detection of barbiturates.
DPC is 0.01% in chloroform. Mercuric sulphate is 0.01% in dilute sulphuric acid.
2,2-Diphenylpicrylhydrazyl For detection of aldehydes and ketones
Dissolve 15mg of 2,2-DPPH in 25ml chloroform
Spray, heat 5-10min at 110C
Yellow spots on a purple background.
Dithizone For detection of heavy metal ions
Dissolve 20mg dithizone in 100ml acetone, store in a brown bottle in a refrigerator
Spray with dithizone solution
Spray with 25% ammonia solution
Dittmer and Lester See Molybdenum blue
Dragendorffs reagent.For detecting alkaloids and quaternary nitrogen compounds
Dissolve 0.11gm potassium iodide and 0.18gm bismuth subnitrate (OBiNO 3) in 20mls acetic acid and
make up to 100ml.
E
Erhlich reagent See p-Dimethylaminobenzaldehyde
Ethanolamine diphenylborate (flavone reagent according to Neu) For detection of flavonoids
1. Dissolve 1gm of ethanolamine diphenylborate in 100ml methanol
2. Dissolve 5gm polyyethylene glycol in 100ml ethanol
Spray with solution 1, then solution 2. Observe at 365nm UV light
Erhlich reagent See p-Dimethylaminobenzaldehyde
Emerson reagent See 4-aminoantipyrine/potassium hexa-cyanoferrate (III)
F
Fast Blue B reagent For detection of cannabinoids, phenols, tanning agents.
Spray with a freshly prepared soution of 0.5g Fast Blue B (tetraazotized di-o-anisidine) in 90ml acetone
and 10ml water.
Then overspray with 0.1M sodium hydroxide solution

Ferric chloride For detecting phenols and phenolic acids.

2.7gm of salt dissolved in 100ml 2M hydrochloric acid.
Ferric Chloride / sulfuric acid Charring reagent for polymer bound TLC plates.
Spray with a solution of 2g FeCl3 in 83ml n-butanol and 15ml conc. sulfuric acid.
Heat at 110C
View at 5 min intervals to see if spots appear at 254 and 360nm.
Heat until spots are brown, grey or black.
Ferric chloride-potassium ferricyanide
Dissolve 3gm of each solid in 100ml 2M hydrochloric acid
For detecting aromatic amines and phenolic compounds.
Flavone reagent according to Neu See ethanolamine diphenyl borate
Fluorescamine
0.005gm of the reagent in 100ml acetone, prepared fresh.
For detecting primary amino acids and amines.
Fluorescamine For detection of primary and secondary amines, peptides, sulfonamides, e.g.,
nitrosoamines after photolysis
Spray plate with a solution of 0.1mg/ml 4-phenyl-spiro[furan-2(3H),1-phthalan]-3,3-dione in 100ml
acetone prepared fresh daily
For stabilization of fluorescence at 360nm spray with 10g triethylamine, made up to 100ml with
dichloromethane.
Fluorescent Indicator For detection of compounds which absorb UV light
Some TLC plates when manufactured have an inorganic fluorescent indicator added to the slurry poured
to make the final plates. This type of indicator will not dissolve off. They are activated at 254nm or
360nm
When activated the fluorescent indicator will turn a green or white and the compounds appear as dark
spots. The compounds might also have some fluorescence of their own, so various colors may also
appear.
Formaldehyde / sulfuric acid For detection of alkaloids, aromatic hydrocarbons, e.g., antihypertensive
drugs
Spray with a solution of 37% formaldehyde (commercial) in conc. sulfuric acid (1:10) as soon as the plate
is removed from the developing chamber.
Various colored spots.
Formaldehyde / phosphoric acid For detection of steroid alkaloids, steroid sapogenins and
phenothiazine derivatives
Spray with a solution of 0.03g formaldehyde in 100ml of conc. phosphoric acid with stirring at room
temperature.
The reagent is stable for several weeks.
Fuchsin reagent For detection of aldehydes
Dissolve 0.05 gm Fuchsin (4-rosaline hydrochloride) in 50 ml water. Add 2 ml saturated sodium bisulphite
solution, leave on bench for 1 hour. Add 1 ml conc. HCl and allow to stand overnight to give a colorless
liquid.
Gives violet to purple spots.
Furfural / sulfuric acid For detection of carbamate esters
Solution I: Dissolve 1gm of furfural in 100ml acetone
Solution II: Dissolve 10ml conc. sulfuric acid in 90ml acetone
Spray with I, then II.
G

Gentian Violet / Bromine For detection of lipids

Spray 0.1gm gentian violet (crystal violet) in 100ml methanol onto plate and place in a tank containing
bromine vapor.
Look for blue spots on a yellow background.
Gibbs reagent For detection of phenols.
Dissolve 3gm of 2,6-dibromo-N-chloro-p-benzoquinone imine in 100ml methanol.
HPT Reagent For detecting bile acids.
Dissolve 0.025gm of 8-hydroxy-1,3,6-pyrenetrisulphonic acid trisodium salt in 100ml methanol.
H
Hydroxylamine / iron (III) chloride For detection of amides, lactones, carboxylic acid esters and
anhydrides
Solution I: Make up a solution of 7g hydroxylammonium chloride in 100ml methanol and a solution of 7.2
g potassium hydroxide in 100ml methanol. Mix both solutions together and filter.
Solution II: Dissolve 2gm of iron (III) chloride and 1ml conc. HCl in 100ml water.
Dry plate in air
Spray with solution I, then with solution II
I
Iodoplatinate For detecting alkaloids, amines, and organic nitrogen compounds.
Dissolve 0.15gm potassium chloroplatinate and 3gm potassium iodide in 100ml dilute hydrochloric acid.
Iodoplatinic acid For detection of alkaloids, cocaine metabolites, amines, analgesics and biotin.
Dissolve 3ml. of chloroplatinic acid (hydrogen hexachloroplatinate) in 100ml. water, then mix with a
solution of 6gm of potassium iodide in 100ml water.
Iron (III) chloride / potassium hexacyanoferrate / sodium arsenate (according to Patterson &
Clements)
For detection of iodine compounds, e..g., thyroid gland hormones
Solution I: Dissolve 2.7gm iron (III) chloride hexahydrate in 100ml of 2N hydrochloric acid
Solution II: Dissolve 3.5gm potassium hexacyanoferrate in 100ml water
Solution III: Dissolve 3.8g arsenic trioxide in 25ml 2N sodium hydroxide solution heating slightly, cool to
5C, and add 50ml 2N sulfuric acid, fill to 200ml with water
Immediately before use mix 5ml solution I, 5ml solution II and 1ml solution III
Isatin For detecting proline, hydroxyproline and other amino acids and peptides.
0.2gm isatin in 100ml methanol.
JKL
Lead tetraacetate/2,7-dichlorofluorescein for detection of vicinal diols, glycosides and phenols, e.g.
sugar acids
Solutions:
1. 2gm lead tetraacetate in 100ml glacial acetic acid
2. 1gm 2,7-dichlorofluorescein in 100ml ethanol
Mix 5 ml each of solution 1 and 2 and make up up to 200 ml with dry toluene. This reagent solution is
stable for only about 2 hours.
M

Mandelin reagent See Vanadium(V) / sulfuric acid

Manganese / salicylaldehyde For detection of organothiophosphorus pesticides
Solution I: Dissolve 100mg manganese chloride (MnCl2.4H2O) in 100ml ethanol.
Solution II: Dissolve 1.3g 2-hydrozine quinoline in a minimum volume of hot ethanol. Dissolve 1 g
salicyaldehyde in 5ml ethanol and add 1-2 drops glacial acetic acid. Combine both solutions and reflux
30 minutes. The crystals of salicyl-2-aldehyde C2-quinolinehydrazone precipitated during the cooling are
recrystallized from ethanol. For solution dissolve 50mg of the salicylate derivative in 100ml ethanol
Spray with a mixture of equal volumes of solutions 1 and 2.
Mercury (II) chloride / diphenylcarbazone For detection of barbiturates
Solution I: Dissolve 2gm mercury (II) chloride in ethanol and make up to 100ml with water.
Solution II: Dissolve 0.2gm diphenlycarbazone in ethanol and make up to 100ml with water.
Mix equal parts of both solutions together just before use.
Mercury (II) chloride / dithizone For detection of barbiturates
Solution I: Dissolve 1.5gm mercury (II) chloride in 100ml ethanol.
Solution II: Dissolve 1.5gm dithizone in 100ml ethanol.
Spray with a mixture of equal volumes of solution I and solution II.
View under 360nm UV light
4-Methoxybenzaldehyde / sulfuric acid / ethanol For detection erythromycin and metabolites
Make up reagent by adding 10ml of 4-methocybenzaldehyde and 10ml sulphuric acid to 90ml ethanol.
Heat 1 minute at 105C
Methyl yellow For detection of chlorinated insecticides and antimicrobial compounds
Spray dried plate with a solution of 0.1g methyl yellow (N,N-dimethyl-4-phenylazoaniline) in 70ml ethanol,
add 25ml water and make up to 100ml with ethanol.
Dry at ambient temperature
Irradiate 5 min with UV light.
Red spots on a yellow background
Molybdatophosphoric acid See under Phosphomolybdic acid
Molybdenum blue reaction according to Dittmer and Lester
For detection of phospholipids and phosphoric acid derivatives
Solution I: Boil 40.11g MoO3 in 1 liter 25N sulfuric acid for 3-4 hours until the molybdenum oxide is
completely dissolved. Let stand at room temperature overnight until solution turns light blue.
Solution II: Boil 1.78 g molybdenum powder and 500ml of solution I for 15 min, cool and decant from the
remaining residue.
Preparation of the spray reagent. Add equal volumes of solutions I and II to 4.5 volume parts water. A
dark green solution is formed.
Solutions I and II are stable for several months when stored in the dark. The spray reagent has to be
prepared weekly.
Molybdenum blue For detecting phospholipids and related compounds.
Dissolve 1.3gm molybdenun oxide and 23.5ml conc. sulphuric acid in 100ml water.
N
Ninhydrin For detection of amino acids, amines, amino sugars(amphetamines).
Dissolve 0.2gm ninhydrin in 100ml ethanol, or in 94ml water and 6ml acetone.
Spray and heat to 110C until reddish spots appear.
Ninhydrin Fixer reagent
Acidified ethanol containing 1% saturated cupric nitrate.
Fixative for ninhydrin chromatograms.
Ninhydrin / cadmium acetate For detection of amino acids and heterocyclic amines

Dissolve 1g ninhydrin and 2.5g cadmium acetate in 10ml glacial acetic acid and fill to 500ml with ethanol.
Spray and heat 20min at 120C
Red, pink, or purple spots are seen.
Ninhydrin / pyridine / glacial acetic acid For detection of peptides
Spray with a solution of 1gm ninhydrin in 95mls pyridine and 5 mls glacial acetic acid.
Heat 5 min at 100 C
Nitric acid / ethanol For detection of amines and alkaloids
Spray with a solution of 50 drops conc. nitric acid in 100ml ethanol. If necessary, heat to 120 C for some
time.
N-4-Nitrobenzyl-N-n-propylamine hydrochloride (NBPA)
0.2 millimole reagent
For detection of isocyanates.
O
1. Orcinol Ferric Chloride (Bials reagent)
For detecting sugars,glycosides, and sulfolipids.
Dissolve 0.9gm ferric chloride and 0.55gm orcinol in 80mls ethanol and 20 mls HCl.
2. Orcinol (Bials reagent)
For detection of glycosides, glycolipids
Dissolve 0.1g orcinol in 40.7ml conc. HCl, add 1ml 1% ferric(111) chloride, and dilute to 100ml
Spray and heat at 80C for 90 minutes.
Glycolipids produce violet spots.
Oxime (8-hydroxyquinoline)
Dissolve 0.25g in 8o ml etanol and 20 ml water
For detection of cations, AL, Cr, Fe, Co, Ni, Mn, Zn.
P
Patterson and Clements See under Iron (III) chloride/potassium hexacyanoferrate/sodium arsenate
Paraffin oil For enhancement of fluorescence spots.
1% paraffin oil in hexane
Spray evenly over the TLC plate
Spots remain stable.
m-Phenylenediamine For detection of reducing sugars
Spray with a solution of 3.6g m-phenylenediamine dihydrochloride in 100ml ethanol and heat briefly at
105C.
Intense fluorescence in UV light.
o-Phenylenediamine - trichloroacetic acid For detection of alpha-keto acids
Spray with a solution of 0.05g 1,2-phenylenediamine in 100ml 10% aqueous trichloroactic acid and heat
plate at 100C for 2 minutes only.
Green fluorescence spots in long wavelength UV light.
p-Phenylenediamine - phthalic acid For detection of conjugated 3-ketosteroids
Dissolve 0.9 p-phenylenediamine and 1.6g phthalic acid in 100ml 1-butanol saturated with waterand heat
plate at 100-110 C
Yellow to orange spots
Phenylhydrazine sulfonate For detection of some antimicrobial compounds
Solution I: dissolve 3.5g phenylhydrazine 4-sulfonic acid hemihydrate in 10ml water and 20ml 1N NaOH
solution

Solution II: mix 30ml 2N sodium hydroxide solution with 40ml acetone
The spray reagents must be prepared fresh.
Wet chromatogram evenly with spray solution 1 and then air dry the plate.
Shake spray solution II and spray plate.
1. Phosphomolydbic acid For detection of reducing substances, e.g, alcohols, bile acids, lipids, fatty
acids, steroids
Spray with a solution of 0.25gm molybdatophosphoric acid in 50ml ethanol
Heat to 120C until spots appear (oven or heat gun)
If necessary, treat with ammonia vapors to remove some background coloration.
The reagent solution is stable for only 1 week even in the dark. Also used as a charring reagent for
polymer bound TLC plates.
When used as a charring reagent, view every 5-10min to observe spots at 254 and 360nm. Spots are
brown, grey or black.
2. Phosphomolybdic acid
For detection of Fatty acid methyl esters, keto acids, lipids, steroids, and sulphated bile acids.
Dissolve 5gm reagent in 100 ml propan-2-ol.
Phosphoric acid For detection of sterols, steroids, and bile acids
Spray heavily until the layer appears transparent with a solution of 50 mls conc phosphoric acid 50 mls
water.
Then heat 10-15minutes at 120C
Phosphoric acid / bromine For detection of digitalis glycosides
Spray solution I: 10% aqueous phosphoric acid solution
Spray solution II: Mix 2ml saturated aqueous potassium bromide, 2ml saturated solution aqueous
potassium bromate and 2ml 25% hydrochloric acid.
1. Spray plate with solution I and heat 12 min at 120C.
Digitalis glycosides of the series B, D, and E show blue fluorescence in long wavelength UV light
2. Heat the plate again at 120C and spray lightly with solution II
Glycosides of the series A show orange, series C show grey-green to grey-blue fluorescence in UV light.
Phosphoric acid / cupric acetate
For detection of Phospholipids.
Dissolve 3gm cupric acetate 10 mls phosphoric acid and 90 mls water.
Phosphotungstic acid For detection of cholesterol and its esters, reducing compounds, lipids, sterols,
and steroids
Dissolve 20gm phosphotungstic acid in 100ml ethanol, heat at 110C for 5-15min or until spots appear.
Cholesterol, esters will give red spots.
Pinacryptol yellow For detection of sweetners, surfactants, alkyl- and arylsulfonic acids
Dissolve 0.10gm pinacryptol yellow in 100ml hot water or ethanol.
Spray and view under UV light.
Yellow to orange fluorescence spots under 366nm. UV lightt.
Potassium dichromate / sulfuric acid (chromosulfuric acid)
Universal visualization reagent for organic compounds (e.g., alcohols, bile acids, lipids)
Dissolve slowly and with stirring 5g potassium dichromate into 100ml conc. sulfuric acid. (Use an ice
bath)
Note: do not use this reagent on polymer bound TLC plates since it will char the binder; use only with
gypsum binder plates.
If necessary heat plate to 150C
Brown, grey, or black spots show up.
Potassium permanganate / sulfuric acid
Universal reagent for organic compounds, e.g. fatty acid derivatives

Note: Do not use this reagent on polymer bound TLC plates since it will char the binder; use only with
gypsum binder plates.
Dissolve slowly and with stirring 1.6gm potassium permanganate into 100 ml conc. sulfuric acid. (Use an
ice bath).
Heat plate for 15-20min at 180C
Pyrocatechol violet reagent for detection of organotin compounds
Completely decompose metalorganic compounds by irradiation of the developed plates with UV light
Dip plates in a solution of 1gm pyrocatechol violet in 1000 ml ethanol (99.5%)
QR
Rhodamine B
For detecting lipids, and
For detecting metals (Au, Bi, Cd, Fe, Hg, Mo, TI, V, and W.)
Dissolve 0.25gm of regent in 100 ml ethanol or acetone.
Placing the sprayed chromatograms into an ammonia atmosphere, increases sensitivity.
Rhodamine 6G
For detecting lipids.
Dissolve 0.01 gm Rhodamine 6G in 100 ml methylene chloride.
1. Rubeanic acid (sodium dithioxide)
For detection of cations gb111A and 111B
Dissolve 1gm reagent in 100 ml ethanol.
2. Rubeanic acid for detection of heavy metal ions
Spray with a solution of 0.5gm rubeanic acid in 100 ml ethanol
Dry briefly
Spray with 25% ammonia solution or place in a chamber with ammonia vapour
S
Silver nitrate / hydrogen peroxide For detection of halogenated hydrocarbons
Make up a solution of 0.1g silver nitrate in 1ml water, add 10ml 2-phenoxyethanol, fill to 200ml with
acetone and add 1 drop hydrogen peroxide (30% solution)
Spray plate and irradiate with UV light. For alumina plates, about 50 minutes, for silica gel plates about
15 minutes
Look for dark spots.
Sodium azide For detection of antibiotics (penicillins and cephlosporins)
Solution I: Dissolve 0.5gm soluble starch 100 ml boiling water and cool down to room temperature.
Solution II: Dissolve 3.5gm sodium azide in 100 ml of 0.1N iodine solution
Spray with solution 1, dry, then spray with solution II
Sodium 1,2-napthaquinone-4-sulfonate (NZS reagent)
For detection of thiazide drugs, basic drugs with primary amino groups
Solution I: 0.1N NaOH
Solution II: saturated solution of reagent in 50 ml ethanol and 50 ml water.
Spray with I, then II.
Thiazide drugs appear as orange spots within 15min; basic drugs with primary amino groups also react.
However, barbiturates do not react.
Sodium nitrite / hydrochloric acid For detection of indoles and thiazoles
Spray plate with a freshly made solution of 1g sodium nitrite in 100ml hydrochloric acid, and heat at
100C.

Indoles turn red and thiazole derivatives turn light green.

Sodium nitroprusside / hydrogen peroxide For detection of guanidine, urea, thiourea and derivatives,
creatine and creatinine.
Mix 2ml of 5% aqueous sodium nitroprusside, 1ml of 10% aqueous sodium hydroxide and 5ml of 3%
aqueous hydrogen peroxide. Dilute to 15 mls. with water.
Sodium nitroprussate / potassium hexacyanoferrate (III) For the detection of aliphatic nitrogen
compounds, cyanamide, guanidine, urea, thiourea and derivatives, creatine, and creatinine.
1. Make up a solution with 15 mls 10% aqueous sodium hydroxide, 15 mls of 10% sodium nitroprussate,
15 mls of 10% potassium hexacyanoferrate (III), and 45 mls of water. Let the solution stand at room
temperature for about half hour before use.
2. Mix the reagent solution with an equal part of acetone and spray.
Stannic Chloride See under Tin (IV) chloride
Sulphuric acid spray reagent
Charring reagent for organic compounds. For detection of bile acids
5% w/v of the acid in ethanol.
T
Tetracyanoethylene - TCNE reagent For detection of aromatic hydrocarbons and heterocycles,
aromatic amines, and phenols
Dissolve 0.5 - 1.0g tetracyanoethylene in 100ml dichloromethane or toluene.
Heat at 100C for a short time.
Tetranitrodiphenyl For detection of cardiac glycosides
Solution I: Saturated solution of 2,3,4,4-tetranitrodiphenyl in toluene
Solution II: Dissolve 10gm potassium hydroxide 50 ml water and 50 ml methanol
Spray with solution I, dry at room temperature, then spray with solution II.
Blue spots are observed.
Tetrazolium blue For detection of corticosteroids and other reducing compounds.
Solution 1. Dissolve 0.5gm tetrazolium blue 50ml water and 50 ml methanol.
Solution II. Make up 100 mls 6M NaOH.
Spray with an equal mixture of both solutions.
Violet spots at room temperature or with slight warming.
Thymol / sulfuric acid For detection of sugars
Spray with a solution of 0.5g thymol in 95ml ethanol, and add 5ml conc. sulfuric acid with caution.
Heat 15-20min at 120C
Sugars show pink spots.
Tin (IV) chloride For detection of triterpenes, sterols, steroids, phenols, and polyphenols
Spray with 10ml tin (IV) chloride in 80 mls of chloroform and 80 mls of glacial acetic acid.
Heat the layer for 5-10min at 100C and inspect in visible and long wavelength UV light.
TNF reagent (trinitrofluorenone) for detection of phenols
Spray with a solution of 2gm 2,4,7-trinitrofluorenone in 100 ml toluene
o-Tolidine, diazotized For detection of phenols
Tolidine solution - Make up 5g o-tolidine and 14ml conc. hydrochloric acid in 100ml water
Nitrate solution - Dissolve 10gm sodium nitrate in 100 ml water, prepared fresh
Mix 20ml tolidine solution and 20ml nitrate solution at 0C stirring constantly.
The spray solution is stable for about 2-3 hours.
Note: After spraying it can take several hours until colored spots are formed.
p-Toluenesulfonic acid For detection of steroids, flavonoids and catechins
Dissolve 20gm of p-toluenesulfonic acid in chloroform and heat a few minutes at 100C.

Inspect under long wavelength UV light.

Trichloroacetic acid For detection of steroids, digitalis glycosides, veratrum alkaloids and vitamin D
Spray solution I: Dissolve 25gm of trichloroacetic acid in 100 ml chloroform.
Spray solution II - for vitamin D - Dissolve 1gm of trichloroacetic acid in 100 ml chloroform.
Spray solution III - for digitalis glycosides - Dissolve 3.3g trichloroacetic acid in 100ml chloroform and add
1-2 drops hydrogen peroxide.
Spray with the appropriate solution, heat 5-10min at 120C and observe in normal light and long UV light.
Inspect the spots in daylight and in long wavelength UV light.
Trifluoroacetic acid For detection of steroids
Spray with a solution of 1gm trifluoroacetic acid in 100 ml chloroform and heat 5min at 120C
Triethanolamine For enhancement of fluorescence species
Dissolve 20gm of reagent in 100 ml isopropanol
Tungstophosphoric acid See under Phosphotungstic Acid
U
Ultraviolet Light - fluorescence Examine the dried chromatogram under both 254 and 360nm UV light.
Ultraviolet Light - quenching For detection of various compounds which absorb 254nm UV light
This wavelength light will appear as dark spots against a green or white fluorescence caused by the UV
activation of the fluorescent indicator in the plate.
Urea / hydrochloric acid For detection of sugars
Spray with a solution of 5g urea in 20ml 2M hydrochloric acid, with 100ml ethanol added and heat to
100C.
Ketoses and oligosaccharides containing ketoses turn blue.
V
Vanadium (V) / sulfuric acid For detection of carbohydrates, glycols, reducing carboxylic acids,
steroids, antioxidants, vitamins, phenols, aromatic amines, antihistamines
1. Spray with a solution of 1.2g ammonium monovanadate in 95ml water and 5ml conc. sulfuric acid.
2. For beta blockers: spray with saturated solution of ammonium monovanadate in conc. sulfuric acid.
Vanadium pentoxide / sulfuric acid Spray with a solution of 1.82g vanadium pentoxide in 30ml 1M
sodium carbonate, sonicate to achieve complete dissolution, after cooling add 46ml 2.5M sulfuric acid
and fill to 100ml with acetonitrile.
Vanillin / potassium hydroxide For detection of amines and amino acids
Spray with a solution of 1g vanillin in 50ml 2-propanol and dry 10min at 110C
Then spray with a solution of 1ml 1M potassium hydroxide added to 100ml ethanol. Dry 10 min at 110C
View at 365nm UV
Vanillin / phosphoric acid Used as a charring reagent for polymer bound TLC plates.
Spray plate with a solution of 1g vanillin in a mixture of 50ml water and 50ml H3PO4
Heat 5-30min at 110C.
View frequently for colored or fluorescent spots (at 254 and 360nm) to appear.
Charring can be continued until spots are brown, grey or black.
Vanillin / sulfuric acid For detection of steroids.
This charring visualization reagent can only be used with glass TLC plates in which a G (gypsum)
binder has been incorporated.
Dissolve igm of vanillin in 100ml conc. sulfuric acid and spray plates. Dry at 120C until maximum color
development.
Another formulation of this reagent: 0.5g vanillin in 80ml sulfuric acid and 20ml ethanol.

WXYZ
None available

Study References:
1. Thin Layer Chromatography, 2nd edition, E. Stahl, ed, Springer-Verlag, NY, 1969
2. TLC Reagents & Detection Methods Physical & Chemical Detection Methods: Fundamentals, Vol 1a,
H. Jork, W. Funk, W. Fishcer, & H. Wimmer, Wiley, NY, 1990
3. Practice of Thin Layer Chromatography, 3rd edition, J.C. Touchstone, Wiley-Interscience, NY, 1992
4. Vogel's Textbook of Qualitative Inorganic Analysis by Vogel, A.I., 3rd, Ed., Longman (1961).

SOLVENTS DRYING AND DRYING AGENTS

Properties of solvents and methods used for drying them with specific drying agents

Drying with sodium wire :

Disposal of spent sodium, magnesium and calcium hydride

REMOVING WATER FROM SOLVENTS

There are various ways of removing water and other impurities from a solution. This can
become a major task once the used reagents are also sensitive towards water e.g. Grignard
reagents or enolates. If water is one of the products, it also has a detrimental effect on the yield
and/or reaction rate. In those cases, drying agents like activated alumina, calcium hydride
(CaH2), sodium metal (in combination with benzophenone which forms a dark blue ketyl radical),
lithium aluminum hydride (LiAlH4) or phosphorous pentoxide (P4O10) are used to chemically
destroy the water.
The drying agents commonly used in the organic laboratories are the anhydrous forms of
calcium chloride (CaCl2), sodium sulfate (Na2SO4) Calcium sulfate (CaSO4 (as Drierite) and
magnesium sulfate (MgSO4).
Organic liquids are considered to be wet if they contain water,but the organic liquid will still be a
liquid after it is dried.
ACTIVATED ALUMINA

Activated alumina is a very porous form of aluminum oxide of high surface area which adsorbs
liquids and gases without any change in form. Activated alumina will not soften or disintegrate
when immersed in liquids. Activated alumina may be regenerated to its original adsorption
efficiency by heating to a temperature between (177-316C).
Activated alumina can be used for:
Reconditioning of mineral oils like transformer or insulating oils.
Removal of oil vapor mist from compressed air.
Drying of organic liquids to a moisture level of 10 ppm or below.
Drying of various liquids such as Benzene, Carbon, Tetrachloride, Chlorobenzene, Ethyl
Acetate, Transformer oil, Vegetable oils etc.

SOLVENTS DRYING GUIDE

TO DRY

USE ONE OF THE FOLLOWING DRYING AGENTS

Alcohols

Anhydrous forms of potassium carbonate; anhydrous magnesium or calcium

sulphate; quicklime.

Alkyl halides
Aryl halides

Anhydrous calcium chloride; anhydrous forms of sodium sulphate,

magnesium sulphate, or calcium sulphate; sodium pentoxide.

Saturated and
Aromatic hydrocarbons

Anhydrous calcium chloride or sulphate; metallic sodium; phosphorus

pentoxide.

Aldehydes

Anhydrous sodium sulphate; anhydrous magnesium or calcium sulphate.

Ketones

Anhydrous sodium sulphate; anhydrous magnesium or calcium sulphate;

anhydrous potassium carbonate.

Organic bases
(amines)

Solid potassium or sodium hydroxide; quicklime; barium oxide.

Organic acids

Anhydrous sodium sulphate; anhydrous magnesium or calcium sulphate.

SOLVENTS: PROPERTIES AND DRYING AGENTS

Solvent

Boiling
Flash
Densit
Pt.
Pt.
y
C
C

Drying agent

Acetone

56

0.791

-18

K2CO3; Molecular sieve 0.3nm;

CaCl2

Acetic acid

118

1.049

+40

P2O5; CuSO4

Acetic anhydride

136

1.082

+49

CaCl2

Acetonitrile

82

0.782

+6

CaCl2; P2O5; K2CO3; calcium

hydride; Molecular sieve 0.3nm

Aniline

184

1.022

+76

KOH; BaO

Anisole

154

0.995

+51

CaCl2; Na; distillation

Benzene

80

0.879

-10

CaCl2; distillation; Na; Pb/Na; ;

calcium hydride; Na wire;
Molecular sieve 0.4nm

1-Butanol

117

0.810

+29

K2CO3; distillation

2-Butanol

100

0.808

+24

K2CO3; distillation

tert-Butanol

82

0.882

+11

CaO; Freezing

n-Butyl acetate

127

0.882

+33

MgSO4;

Carbon disulphide

46

1.263

-30

CaCl2P2O5;

Carbon tetrachloride

77

1.594

none

distillation; P2O3; Pb/Na;

Molecular sieve 0.4nm

Chlorobenzene

132

1.106

+29

CaCl2; distillation;P2O5

Chloroform

62

1.486

none

CaCl2; P2O5; Pb/Na; Molecular

sieve 0.4nm

Cyclohexane

81

0.799

none

Na; Na/Pb; LiAlH4; Molecular

sieve 0.4nm

Cyclohexanone

155

na

na

Distillation

Decahydronaphthalene
(Dekalin)

~190

0.886

<54

CaCl2; Na; Pb/Na

Dichloromethane

40

1.325

none

CaCl2; Pb/Na; calcium hydride;

Molecular sieve 0.4nm

Dicyclopentadiene:
(cyclopentadiene dimer)

170

na

na

Refractionation: distillate at 40 42C. (Use at once! or keep in

dry ice/acetone bath no longer
than 1 Hr.)

Diethyl carbonate

126

0.975

+25

Na2SO4; K2CO3

Diethylene glycoldibutyl ether

225

0.885

+118

CaCl2; Na

Diethylene glycol dimethyl

ether

155
165

0.906

+70

CaCl2; Na

Diethyl ether

34

0.714

-40

CaCl2; Na; Pb/Na; LiAlH4; Na

wire/benzophenone; Molecular
sieve 0.4nm

Di-isopropyl ether

68

0.726

-23

CaCl2; Na; Molecular sieve

0.4nm

Dimethyl formamide

153

0.950

+62

distillation; Molecular sieve

0.4nm

Dimethyl sulfoxide

189

1.101

+95

distillation; Molecular sieve

0.4nm

1,4 Dioxane

101

1.034

+11.8

CaCl2; Na; Molecular sieve

0.4nm

Ethanol

79

0.791

+12

CaO; Mg; MgO; Molecular sieve

0.3nm

Ethyl acetate

77

0.901

-4

K2CO3; P2O5; Na2SO4; calcium

hydride; Molecular sieve 0.4nm

Ethylenediamine:
(1-2 diaminoethane)

118

na

na

Simple distillation.

Ethylene glycol

197

1.109

+111

distillation; Na2SO4

Ethylene glycol monoethyl

135

0.930

+41

distillation

ether
Ethylene glycol monomethyl
ether

125

0.965

+52

distillation

Ethyl formate

54

0.924

-20

MgSO4; Na2SO4;

Formamide

211

1.134

155

Na2SO4; CaO

Glycerol

290

1.260

+176

distillation

Heptane

98

0.684

-4

calcium hydride; Na wire

n-Hexane

69

0.659

-23

Na; Pb/Na; LiAlH4; calcium

hydride; Na wire/benzophenone;
Molecular sieve 0.4nm

Isobutanol

108

0.803

+28

K2CO3; CaO; Mg;

Isobutyl methyl ketone

117

0.801

+15.5K2CO3;
4

Methanol

65

0.792

+11

Mg; CaO; Molecular sieve 0.3nm

Methyl acetate

57

0.933

-10

K2CO3; CaO;

1-Methyl-2-pyrrolidone

202

1.026

+95

Na2SO4; distillation; Molecular

sieve 0.4nm

Methyl Ethyl ketone

80

0.806

-44

K2CO3;

Nitrobenzene

211

1.204

+92

CaCl2; P2O5; distillation;

n-Pentane

36

0.626

-49

Na; Pb/Na;calcium hydride; Na

wire

Pet ether

mixture na

na

calcium hydride; Na
wire/benzophenone; Molecular
sieve type 4A

1- Propanol

97

0.804

+15

CaO; Mg

2-Propanol

82

0.785

+12

CaO; Mg; Molecular sieve 0.3nm

Pyridine

116

0.982

+20

KOH; BaO; Molecular sieve

0.4nm

Tetrahydrofuran

66

0.887

-17.5

Molecular sieve 0.4nm

Tetrahydronaphthalene
(Tetralin)

208

0.973

Thionyl chloride

48

na

na

Redistill.

Toluene

111

0.867

+4

distillation; Ca; CaCl2; Na;

Molecular sieve 0.4nm

Trichloroethylene

87

1.462

none

distillation; Na2SO4; K2CO3

Xylene

137/140 -0.86

+25

distillation; Na; CaCl2; Molecular

sieve 0.4nm

CaCl2; Na

Reference: http://www.deltaadsorbents.com/activated_alumina_2.php

STANDARD SIEVES AND MESH SIZES

SIEVES STANDARD NO. MESH SIZES AND
DESIGNATIONS
See below for Particle size conversions

Mesh Size
(microns)

TYLER

ASTM-E11

BS-410

DIN-4188

Mesh

No.

Mesh

mm

2500

2500

0.005

10

1250

1250

0.010

15

800

800

0.015

20

625

625

0.020

22
25

0.022
500

500

0.025

28

0.028

32

0.032

36

0.036

38

400

400

400

40
45

0.040
325

325

350

50
53
56

0.045
0.050

270

270

300
0.056

QUALITATIVE ANALYSIS LABORATORY REAGENTS

This page gives info on the preparation of the laboratory test reagents and solutions
used in qualitative analysis. The bench reagents consist of a stack of acids and bases on
each bench top. Side bench and Special reagents are placed away from the work bench
on reagent shelves. A lab will only put out those reagents that are necessary for the
determinations being carried out at the time.

THE PH COMBINATION ELECTRODE

This page gives info on the different types of pH combination electrodes, their uses, operation,
care, maintainance,
reconditioning and glass membrane cleaning, as well as electrode and meter troubleshooting.
COMBINATION PH ELECTRODES
Use electrode combinations that are compatible with the solutions to be measured.
General purpose combination electrode
Has silver chloride wire and KCl electrolyte. Refillable.
Used for aqueous solutions of compounds that do not react with silver.
Must not be used in solutions of :
1. Heavy metals
2. Proteins
3. Low ion solutions (DI water)
4. High sodium
5. Sulphides
6.Tris buffers
7. Organics.
If sample contains any of these contaminants, the pH electrode may work for a short time and
will then fail to operate.
Reconditioning may restore it for a while, but the same short term usage may occur.
Non-refillable type
Permanently sealed with gel fill solution.
Rugged, but has a shorter life, easy to use and inexpensive.
Calomel combination electrode
Has KCl wire and mercuric chloride, with a KCl reference electrolyte. No silver wire. Refillable.
Fill solution is less reactive, does not react with sample, but is temperature sensitive.
Designed to work in solutions containing proteins, organics, low ion activity, tris buffers and
heavy metals.
Double junction electrode
Two chambers. Upper chamber has silver chloride wire/KCl saturated with AgCl, and lower

chamber has a KNO3 electrolyte solution.

Designed to work in same applications as for the calomel electrode, but at higher concentrations
of problem materials.
Teflon junction electrode
Has AgCl wire and porous teflon liquid junction.
Designed so that solutions being measured do not clog the liquid junction.
Used for substances such as oils, foods, paints, gells, and pastes.
Solid-state electrode
Uses FET (Field effect transistor) sensitive to pH.
No glass bulb, non-glass sensor, zero maintainance, but proper electrode practice should be
observed.
MAKING PH MEASUREMENTS
1. Remove fill hole cover when measuring. Fill hole must be open to avoid contamination of
electrolyte from buffer solution.
2. Use ATC. pH is temperature sensitive.
3. Stir solutions using magnetic stir bar / stir plate.
4. Insulate stirrer base and sample container with piece of cardboard or styrofoam to minimize
heat transfer.
5. Use fresh buffer solutions when calibrating meter.
6. Rinse electrode between measurements with distilled water, then with next sample to be
measured.
7. Do not rub electrode glass bulb. Simply, touch-dry with tissue.
8. Never store pH electrode in distilled or deionized water because the filling solution will leak
out into it.
9. Store pH electrode in pH 7.00 buffer with a drop or two of saturated KCl solution added.
For long term storage, fill electrode chamber with filling solution, cover the fill hole, then add a
few drops of saturated KCl into the protective plastic cap and cap the electrode.
10. Do not touch or move the electrode cable while taking a measurement, because readings
may become unstable due to the high impedence of the pH glass membrane.
RECONDITIONING AND CLEANING OF PH ELECTRODES
Poor response of pH electrodes. Prolonged use and natural aging, excessive alkaline
immersion and high temperature operation will cause leaching of the membrane glass and a
reduction in the Nerstian response, which will give rise to noisy and erratic or slow and sluggish
electrode readings. There are twwo causes for this :
1. Contamination of the glass membrane.
2. Clogging of the liquid junction.
The following procedures will often provide renewed stability and pH sensitivity.

Cleaning the glass membrane

(a) Immerse electrode tip in 0.1M HCl for about 15 secs., rinse with distilled water, then immerse
in 0.1M NaOH for another 15 sec.
Cycle the electrode through these solutions several times, then check for performance in pH
buffer 4.00 and 7.00.
(b) If problem still exists, immerse electrode tip into a 20% ammonium bifluoride for 30 secs., or
a 10% hydrofluoric acid solution for 15 secs.
Throughly rinse the electrode with distilled water, then immerse tip into conc. HCl for 30 secs. to
remove residual fluorides, then rinse again with distilled water.
Soak electrode in pH 4.00 for 1 hour, then recheck electrode performance. If not restored,
replace the electrode.
Other methods of reconditioning :
(a) Soak in 0.1M HCl or HNO3 for 1 hour.
Do a second cleaning : soak in 1 : 10 dilution bleach in a 0.1 - 0.05 % liquid detergent in hot
water with vigorous stirring for 15 mins.
(b) soak in saturated KCl @ 50 C for 1 hour. Allow KCl solution to cool down to room
temperature, then rinse with deionized water.
This should clear all contaminants.
Removal of deposits
Protein : Soak in 1 % pepsin in 0.1M HCl for 15 mins.
Inorganic : Soak in 0.1M tetra sodium EDTA solution for 15 mins.
Grease and oil : Rinse electrode in mild detergent in methanol solution.
Unblocking the liquid junction
Inspect reference chamber for cryslallization. Remove crystals as follows :
1. Shake out filling solution from fill hole and flush and rinse reference cavity with distilled water
until all crystals are dissolved.
2. Empty filling cavity and fill up with filling solution.
3. Pressurize the electrode and establish flow :
Insert the spout of the filling solution plastic dispensing bottle into the fill hole of the electrode to
make an air tight seal, then squeeze bottle to exert a pressure into the electrode chamber. A
bead of liquid should form at the liquid junction in about 30 secs. It may be necessary in some
cases to maintain the pressure for several minutes.
If still no flow, soak tip in conc ammonia solution for 5 - 10 mins. (hood), rinse with deionized
water and apply pressure to filling hole.
For gel-filled electrodes:
Soak electrode in water @60 C for 5 - 10 mins. or

Place electrode in warm saturated KCl @ 60 C and allow electrode and solution to cool to
room temperature.
ELECTRODE TEST
1. Calibrate meter with pHbuffer 7.00 according to meter instructions.
Then measure mv for pH 7.00. It should read 0.0 mv 20mv. If reading is greater than this, the
electrode should be reconditioned or replaced.
2. Perform a calibration with buffer pH4.00 or 10.00. Then measure mv reading of the buffer.
Reading should be between 160 - 180 mv. If value is greater than 12 mv of 177 mv, the
electrode should be reconditioned or replaced.
Note :Actual mv values may change as electrode ages, but mv difference will remain between
160 and 180 mv.
METER TEST
Perform pH 7.00 calibration according to meter instructions. If you cannot calibrate to pH 7.00,
you may have a bad electrode.
Now, disconnect the electrode and short out the meter using shorting strap or pin. With the input
shorted out, the mv reading should be 0.0mv, equivalent to pH 7.00.
If out of this range by 2 mv, the meter is bad and should be electronically calibrated.
If you cannot adjust to pH 7.00 with the input shorted out, there is a problem with the meter.
If you can calibrate to pH 7.00 with the input shorted out, the electrode is bad and should be
reconditioned or replaced.
MEASURING ELECTRODE SLOPE
New electrodes have a slope between 95 % and 102 %.
Determine the mv value of pH 4.00 and pH 7.00 buffers, and determine the net change in
millivolts.
e.g. if pH 4.00 = 159.1 mv
and pH 7.00 = -10.0 mv
then net change = 169.1 mv.
Now, divide net change by 177.5, [177.5 mv is the ideal output at pH 4.00 (59.16 X 3 =177.5)]
then multiply by 100 to determine the % of the electrode slope.
i.e. 169.1 / 177.5 x 100 = 95.3 %
If slope remains below 90.0 % or above 102 % after cleaning and /or reconditioning, replace the
electrode.

References :
1. Ag / AgCl reference electrode manual. - Orion research
2. pH Combination electrode instructions. - Fisher Accumet

CLASS OF LABORATORY FIRES AND FIRE

EXTINGUISHERS
Different types of fires and the extinguishers approved to fight them.
Rating, classification, and uses of fire extinguishers.

CLASSIFICATION OF FIRES
Fires are classified according to the materials they burn. They are coded with the capital letter of
the alphabet A, B, C, D, F, etc.. with the word 'Class' placed in front of each letter. Example
Class A fires, Class B fires, Class C fires etc..
Class A Fires Fires involving combustibles such as : wood, paper, boxes, plastic, packing
material etc..
Class B Fires Ignition of flammable liquids such as : solvents, kerosene, gas, grease etc..
Class C Fires Fires arising from electrical equipment such as : AC outlets, wiring, appliances,
flammable gases etc..
Class D Fires Combustible metal fires such as : Mg, K, Na, Al, Titanium, Lithium (includes
powders and swarfs).
Class E Fires Electrical fires : Fires involving electrical apparatus.
Class F Fires Fires involving coking oils and Fats : burning hot oil, cooking oil, lard.
Class K Fires Fires in cooking utensils and appliances caused by oils and fats.
Note

There is no class E or class F fires in the US system of classification. Class E is

equivalent to class C and class F is equivalent to class K.

Class E is no longer applicable because when power is turned off, electrical fires can fall
into any category.

CLASSIFICATION OF FIRE EXTINGUISHERS

Fire extinguishers are classified according to 'class' of fires they are made to put out. An ABC
extinguisher can be used to put out a Class A, Class B, or Class C fire.
An AB extinguisher is used to out a Class A or Class B fire.
And a D extinguisher can be used to extinguish a Class D fire only.
FIRE EXTINGUISHER COLORS

SAFETY IN THE CHEMISTRY LABORATORY

SAFETY MANAGEMENT GUIDELINES

Safety demands an awareness of your surroundings. Be alert to

unsafe conditions or actions in the laboratory. Call attention to them
and make corrections.

Have an evacuation plan and be familiar with it. Keep passageways

clear. Employ exits.

Learn to interpret MSDS info in regard to health hazard, flammability,

reactivity, storage and disposal. Check for TLV, IDLH, Flash point and
Fire fighting media.

Know how to use laboratory safety equipment and the exact positions
of safety shower, eye wash station, respiratory gear, fume hood, fire
alarm, fire extinguisher, and spill clean up materials.

Be aware of ignition sources, open flames, heat and electrical

equipment.

LABORATORY SAFETY PRECAUTIONS

Laboratory Clothing.
Wear shoes that fully cover the feet. Sandals and clogs are not adequate.
Shoes provide a great deal of initial protection in the case of dropped
containers, spilled chemicals, and unseen hazards on the floor. Use old
clothes, which are not too loose, especially at the sleeves. Laboratory coats
or aprons must be worn over clothes. Snaps or fasteners are preferable to
buttons for quicker removal in case of an emmergency. Tie back long hair so
that it will not fall into flames or chemicals.
Avoid shorts and mini skirts in the lab. Exposed body skin give added risk to
irritation and burns by corrosive chemicals and gases.
Aprons
Plastic or Rubber - protects against corrosive and irritating chemicals.
Labcoats
Cotton - good against flying objects, sharp or rough edges (usually treated
with a fire retardant)
Wool - protects against molten splashes, small acid spills and small flames.
Synthetic fibres - protects against IR and UV radiation but burns easily and
can be ruined by strong solvents.
Safety Glasses.
Wear safety glasses at all times in the laboratory. Goggles are required to be
worn at each lab period and should also be worn over prescription glasses.
Contact lenses should not be used during the lab. Goggles designed for
contact wearers should be made available.
Working Alone in the laboratory
All work must be performed under the supervision of a laboratory
instructor/demonstrator. The instructor should be aware of the exact nature
of all work being done in the laboratory.

STORAGE OF INCOMPATIBLE CHEMICALS

This list includes only the most important examples. These chemicals react
violently when they come into contact with each other and must not be stored
together.Mixing incompatible chemicals in a waste container can form an
explosive mixture, for example, nitric acid and ethanol. If a bottle broke in a
waste storage area where incompatibles were present, the results could be
disastrous. Remember: incompatible bottles of wastes should be stored
separately. The objective is to avoid accidents in the laboratory.
See table below for the possible reactions that may occur due to impropper
storage of chemicals.

INCOMPATIBILITY TABLE

Substance

Incompatible with

Acetic acid

Chromium oxide, nitric acid, perchloric acid,

peroxides, permanganates, alcohol, ethylene
glycol

Acetic anhydride

Hydroxyl-containing compounds e.g. ethylene

glycol, perchloric acid

Acetone

Concentrated nitric acid and sulphuric acid

mixtures, hydrogen peroxide

Acetylene

Chlorine, bromine, fluorine, copper, silver,

mercury

Activated carbon

Calcium hypochlorite, oxidizing agents

Water, carbon tetrachloride and other

CHANGING THE HPLC COLUMN FRIT

BACKFLUSHING THE HPLC COLUMN
Caution
In general, it is worth trying to clean and back flush an HPLC column with
appropriate solvents before trying to replace the frit. There is always a danger that
small amounts of packing material may be lost when a frit is replaced and column
efficiency will decrease. In addition, the use of an 0.5 m in-line filter to capture
particulate material and routine column washes with an appropriate strong solvent
are highly recommended.
Symptoms of column plugging
When a column becomes plugged, chromatographic performance generally suffers.
System back pressure increases and, in some instances, peaks become distorted or
split, as well.
Back Flush First
When plugging of the inlet frit occurs there are two ways to restore column
performance. Bach flush or change frit. The easiest and fastest way is to back flush
particulates off the inlet frit.
Back-flushing the inlet frit:
1. Connect the column to the chromatograph so that the flow now is in the reverse
direction. Do not connect the column to the detector because dislodged particulates
from the inlet frit may flow into the detector flow cell.
2. Back-flush the column with first with 100% HPLC grade water at a flow rate of
0.5ml per minute for 2 to three hours. Then repeat at the same rate with 100% HPLC

Acetonitrile. If this does not work, try flushing with mix of 50% methanol and 50%
water.
3. Connect the column to the chromatograph so that the flow is in the proper
direction.
4. Check to see if chromatographic performance is acceptable.
If back flushing the column or column regeneration does not solve the problem,
follow this procedure to replace the inlet frit.

HOW TO REPLACE THE COLUMN INLET FRIT:

1. Place the column in a ring stand or a bench vice with the column inlet pointing up.
2. Use a marking pen to mark the position of the end fitting relative to the column
nut. A vertical mark directly across the end fitting and column nut is recommended.
When you tighten the end fitting after replacing the inlet frit, these marks can help
you torque the nut and end fitting back to the same position.
3. Allow the column to come to room temperature before removing the end fitting. Do
not hold the column while replacing the frit. Heat from your hand can cause
expansion of solvent in the column and may extrude packing material from the open
end of the column.
4. Hold the column end fitting steady with one wrench while loosening the column
nut counterclockwise with another wrench until the nut drops away from the end
fitting. Lift the end fitting off the column.
5. Remove the column inlet frit by carefully sliding it to the side. If the frit is lodged in
the end fitting, remove it by tapping it on a lab bench until the frit drops out.
6. Clean packing material from the top of the column by gently scraping across the
top with a razor blade or a flat spatula. Be careful not to disturb the packing bed
inside the column.
7. Place a new frit on the newly cleaned column inlet.
8. Remove all residual packing material from the column end fitting, tube ferrule, and
column nut. This is easily done by flushing with isopropanol. A squirt bottle works
well for this purpose.
9. Replace the column end fitting and tighten the column nut l/s to 1/4 turns past
finger tight. Align the vertical marks that were placed on the end fitting and column
nut in Step 2 so that the nut is tightened back to the proper torque.
10. Reconnect the column to the chromatograph.
11. Start flow through the column and check for leaks. If the column leaks, turn the

flow off, allow the pressure to bleed off, and then tighten the nut slightly more. If the
column still leaks, some residual packing material probably remains on a sealing
surface (column ferrule, end fitting, threads of the column nut). Remove the end
fitting again and reclean the surfaces.

REFERENCE:
1. MAC-MOD Analytical Inc.

HPLC TROUBLESHOOTING GUIDE

COLUMN LEAKS, RETENTION TIME, BACK
PRESSURE, PEAKS AND BASELINE PROBLEMS

START UP - PRELIMINARY CHECKS

Problem

Possible cause

Solution

Detector off

Check detector

Broken
connections to
recorder

Check connections

No peaks or
very small
No sample/Wrong Check sample. Be sure it is not
peaks
sample
deteriorated. Check for bubbles in the vials
Wrong settings on
recorder or
Check attenuation. Check gain
detector
Pump off

Start Pump

Flow interrupted

Check reservoirs. Check position of the

inlet tubing. Check loop for obstruction or
air. Check degasing of mobile phase.
Check compatibility of the mobile phase
components.

Leak

Check fittings. Check pump for leaks and

precipitates. Check pump seals.

Air trapped in the

system

Disconnect column and prime pump. Flush

system with 100% methanol or
isopropanol. Contact servicing if
necessary.

No Flow

COLUMN AND FITTINGS LEAKS

PREPARATION OF PH BUFFER SOLUTIONS

PHOSPHATE AND ACETATE BUFFERS

The different names for phosphate salts. Standardization buffers pH 4 and pH 7. Ph
range of some buffer systems. Making up buffer solutions by adding an adjuster
solution (acid or base) to a known volume and concentration of a primary salt solution.
Potassium hydrogen phosphate, potassium dihydrogen phosphate, disodium
hydrogen phosphate, potassium hydrogen phthalate, sodium acetate,sodium
tetraborate, tris aminomethane.

PHOSPHATES
Phosphate salts are known by several names and the correct phosphate must be used to
prepare buffer solutions.
One phosphate cannot be substituted for another phosphate. Check formula of salt to be
certain.
Formula

KH2PO4

K2HPO4

K3PO4

Name of salt

Other names

potassium dihydrogen
phosphate

potassium dihydrogen orthophosphate

monobasic potassium phosphate
monopotassium phosphate
acid potassium phosphate
potassium biphosphate

potassium hydrogen
phosphate

dipotassium hydrogen orthophosphate

dipotassium hydrogen phosphate
dibasic potassium phosphate
dipotassium phosphate

potassium phosphate

tribasic potassium phosphate

tripotassium phosphate

STANDARDIZATION BUFFERS
For pH=7.00 :
Add 29.1 ml of 0.1 molar NaOH to 50 ml 0.1 molar potassium dihydrogen phosphate.

Kjeldahl Nitrogen Determination Apparatus

OPERATION OF THE GERHARDT KJELDAHL
N - Determination Apparatus
1. Sample Digestion
a. Weigh required amount of sample onto a clean dry watch glass. Use a
rough balance for this purpose.
b. Weigh sample and watch glass to the nearest mg on an analytical balance.
c.

Transfer the sample into a clean dry digestion tube. and reweigh the watch
glass. Find the mass of sample trassfered.

d. Add to each sample one catalyst tablet and 10 ml conc. sulphuric acid.
(Care! Use dispensette set up in fume hood.)
e. Place sample tubes into digestion heating block, put vapor removal manifold
in place, and turn on water vacuum pump.
f.

Set heater to 400 and allow to digest until sample completely dissolves to
give a clear light green to light blue solution. (1 - 2 hours @ 400) Shut off
heater.

g. Lift sample rack and vapor removal manifold clear off the heating block,
suspend on holding slot, and allow to cool to 35 - 40C.
Switch off heating block, and turn off water vacuum pump when samples are
cooled.

2. Ammonia Distillation and Titration

a. Place respective feed tubes into their reagent resevoirs: 0.1M HCl; 40%
NaOH; 1% w/v Boric acid; and Distilled water supply for steam distillation.
Clamp or wedge feed tubes to avoid their jumping out of the resevoirs during
reagent feed.
b. Remove and empty titration bowl by carefully turning the bowl through 90%
and moving vertically downwards. Rinse electrodes and chamber with
distilled water and replace.
c.

Switch on instrument and turn on water supply (from mains) to condenser.

d. Reply "yes" to prompt for steam distillation.

e. Reply "yes" to prompt for burette flush. Allow syringe to fill and empty twice
and press STOP when syringe is filled for a third time.
f.

Press Boric acid switch and hold down for 5 - 6 seconds. Titration chamber

should then be charged (filled) with boric acid.

g. Depress spring-loaded lever on LHS of instrument while holding the sample
tube in the steam distillation port. Remove the sample tube carefully,
ensuring that the steam inlet tube is not bent when the lever is released.
Replace with a tube containing a sample and ensure that it is properly
seated and sealed when the lever is released.
h. Calibration of electrode.

Press "cal". Select pH by pressing"no" until pH flashes, then press"yes"

Select "calbr". Instrument will now ask for pH buffer 7.0. Place buffer in a ~
20 ml glass container vial to immerse sensing electrode. Avoid contacting
the stirring paddle during this operation. Press "run".

After reading is stabilized, press "Ent". Instrument then asks for pH4.0
buffer. Rinse electrode with distilled water and immerse in pH 4.0 buffer in a
vial. Press "run" and wait for reading to stabilize. The readout will indicate
accecptability of calibration, and the slope of the electrode response.

3. Entering molarity of titrant HCl.

a. Press "cal" and select "titrant" then "keyboard".
b. Enter value of titrant and press "Ent"

4. Determination of % recovery
a. Press "cal" and select "test"
b. Weigh and transfer accurately an ammonium salt of known purity and
composition e.g. ( 0.05g NH4Cl or 0.01g (NH4)2SO4) into a sample tube,
which is then placed in the steam distillation port. Enter theoretical % N
(26.17% for NH4Cl and 21.19% for (NH4)2SO4).
c.

Press "run". Steam distillation will start and end, dependent on program
used. (Use program #6)

d. At the end of the distillation, the sample tube followed by the titration
chamber, will be autumatically emptied, and the latter recharged (refilled).
e. The % recovery of the sample will be displayed.

5. Determination of N-content of samples

a. Place sample tube (with digested sample) in steam distillation port.

b. Press "sample". Press #6 when program # prompt is displayed.

c.

Enter sample number (1, 2, etc....).

d. Enter sample weight.

e. Press "run". Distillation/titration ensues, at the end of which % N for sample
is displayed. Record value displayed.
f.

Replace sample tube with second sample and continue as before.

6. Shutting down of instrument

a. After all samples have been analyzed, carefully remove the feed tubes from
their respective resevoirs, and cap each resevoir securely.
b. Rinse the outsides of the feed tubes with distilled water, and then place
them into a 500 ml Erlenmeyer flask filled with distilled water.
c.

Switch off the instrument for about 5 seconds, and then ON again. Follow
steps 2 (b - f).

d. Follow steps 5 (b - e), entering fictitious values for (d). Stop

distillation/titrations after about 1 minute.
Press "emty" to empty sample tube.
e. Remove sample tube, and rinse steam inlet tube with distilled water.
Replace sample tube in distillation port.
f.

Switch off instrument and turn off water supply to condenser.

250ML VOLUMETRIC ANALYSIS STANDARDS AND

SOLUTIONS
PREPARATION OF 0.1M AND 0.1N VOLUMETRIC ANALYSIS TITRATION
STANDARDS
This page gives info on the weight of salt required to make up 250 ml volumetric
analysis standards for titration against acid base, permanganate, dichromate, iodine,
sodium thiosulphate and silver nitrate. Concentrations are expressed in molarity,
normality and equivalents.

ACID / BASE TITRATIONS

Example for the info on this page:

Sodium carbonate
Na2CO3, FW = 106, Eq. =53g/l
250ml 0.05M = 1.325g = 0.1N

In this example, the FW or Formula weight (Molecular weight) of the salt is 106. Eq. is the
equivalent weight which is 53 grams per liter. And 1.325 grams dissolved up to mark in 250
ml volumetric flask will give a 0.05 M standard solution or a 0.1N standard solution.

Ammonium sulphate
(NH4)2SO4. FW = 132.14, Eq. = 66g/l.
250ml 0.05M = 1.325g = 0.1N

Adipic acid
HO2C(CH2)4CO2H, FW = 146.4, Eq. =
73.03g/l
250ml 0.05M = 1.83g = 0.1N

Barium hydroxide
Ba(OH)2.8H2O, FW = 315.48, Eq. = 157.5g/l
250ml 0.05M = 3.94g = 0.1N

Benzoic acid
C6H5COOH, FW = 122.12, Eq. = 61g/l
250ml 0.05M = 1.52g = 0.1N

Calcium carbonate
CaCO3, FW = 100.00, Eq. = 50g/l
250ml 0.05M = 1.25g = 0.1N

Furroic acid
FW = 112.08, Eq. = 112g/l
250ml 0.1M = 2.8g = 0.1N

Hydrochloric acid
HCl, FW = 36.5, Density = 1.2
1M = 83mls = 1N (Use 86mls)
250ml 0.1M = 2mls = 0.1N
1 liter 0.1M soln = 8.6mls = 0.1N

Oxalic acid
H2C2O4.2H2O, FW = 126.07, Eq. = 63g/l
250ml 0.05M = 1.575g = 0.1N

Potassium hydrogen phthalate

KH(C8H4O4), FW = 204.23, Eq. =204g/l
250ml 0.1M = 5.105g = 0.1N

Potassiun hydrogen iodate

KH(IO3)2, FW = 389.92, Eq. 73.07g/l
250ml 0.1M = 9.75g = 0.1N

Sodium carbonate
Na2CO3, FW = 106, Eq. =53g/l
250ml 0.05M = 1.325g = 0.1N

Sodium hydroxide
NaOH, FW = 40, Eq. = 40g/l
250ml 0.1M = 1.0g = 0.1N
1 liter 0.1M soln = 4g = 0.1N

Sodium oxalate
Na2C2O4, FW = 134.00, Eq. =134g/l
250 0.1M = 3.35g = 0.1N

Sodium tetraborate
NaB4O7.10H2O, FW = 381.37, Eq. =190g/l
250ml 0.05M = 4.762g =0.1N

Succinic acid
HO2CCH2CH2CO2H, FW = 118.09, Eq. =
59.045g/l
250ml 0.05M = 1.475g = 0.1N

Sulphamic acid
H2NSO3H, FW = 97.09, Eq. = 99.09g/l
250ml 0.10M = 2.425 = 0.1N

Sulphuric acid
H2SO4, FW = 98.08, Density = 1.8
1M = 56mls = 2N
250ml 0.05M = 0.7mls = 0.1N
1 liter 0.05M = 2.8mls = 0.1N (use 3 mls)

Tris (hydroxymethyl)-aminomethane
H2N.C(CH2OH)3, FW = 121.14, Eq. =
121g/l
250ml 0.1M = 3.023g =0.1N

TITRATIONS WITH PERMANGANATE

5Na2C2O4 + 2KMnO4 + 8H2SO4 = 10CO2 + 2MnSO4 + 5Na2SO4 + 8H2O

Reaction of Potassium permanganate and Sodium oxalate

Ferrous ammonium sulphate

FeSO4(NH4)2SO4.6H2O, FW = 392, Eq.
392g/l
250ml 0.1M = 9.8g = 0.1N (add a few
drops of conc. H2SO4 to clear)

Ammonium oxalate
(NH4)2C2O4, FW = 124, Eq. = 62g/l
250ml 0.05M = 1.55g = 0.1N

Hhdrogen peroxide
H2O2, FW = 34. Eq. = 17g/l
10 Vol. = 3%
20 Vol. = 6% = 1.8M
100 Vol. = 30%
250ml 0.05M = 12.50ml of 20 Vol. = 0.1N
1 liter 0.05M = 50ml of 20 Vol. = 0.1N (add 2 mls
conc. H2SO4 per liter soln)
1 liter 0.05M = 10mls of 100 Vol.
1 liter 0.05M =35mls of 30 Vol.
Volume strength = 11.2/34 x strongth of peroxide
in g/l

Potassium permanganate
KMnO4, FW =158.04, Eq. ==31.6g/l
250ml 0.02M = 0.79g = 0.1N (dissolve in hot
water)
1 liter 0.02M = 3.16g = 0.1N (boil to dissolve
crystals, then dilute to 1liter)

Iron allum (ferric ammonium

sulphate)
FeNH4(SO4)2.12H2O, FW = 482.19,
Eq. =241g/l
250ml 0.05M = 6.02g = 0.1N
Oxalic acid (anhyd.)
H2C2O4, FW = 90, Eq. = 45g/l
250ml 0.05M =1.125g = 0.1N

Sodium nitrite
NaNO2, FW = 69.00, Eq. = 34.56/l
250ml 0.05M = 0.86g = 0.1N (use 1g)
Sodium oxalate
Na2C204, FW = 234, Eq. = 67g/l
250ml 0.05M = 1.675g = 0.1N

TITRATIONS WITH DICHROMATE

K2Cr2O7 + 6Fe(NH4)2(SO4)2 + 7H2O4 = 3Fe2(SO4)3 + Cr2(SO4)3 + K2SO4 + 6(NH4)2SO4 +

7H2O
Reaction of Potassium dichromate and Ammonium ferrous sulphate

Potassium dichromate

Tin, Sn,

K2Cr2O7, FW = 294, Eq. = 49g/l

250ml 0.0167M = 1.22g = 0.1N
1 liter 0.0167M = 4.90g = 0.1N
Potassium iodate
KIO3, FW = 214, Eq. = 35.73g/l
250ml 0.0167M = 0.892g = 0.1N

FW = 119.0, Eq. = 59.5g/l

Tin (11) chloride (use metallic tin dissolved
in conc. HCl)
250ml 0.05M = 1.49g = 0.1N
Spathic iron ore
FeCO3, FW = 116, Eq. = 116g/l
250ml 0.1M = 2.9g = 0.1N

TITRATIONS WITH IODINE AND THIOSULPHATE

I2 + 2Na2S2O3 = Na2S4O6 + 2NaI

Reaction of Iodine and Sodium thiosulphate

Sodium thiosulphate
Na2S2O3.5H2O, FW = 248.18, Eq. = 248g/l
250ml 0.1M = 6.2g = 0.1N
1 liter 0.1M = 24.8g = 0.1N

Potassium iodide
KI, FW = 166, Eq. =166g/l
250ml 0.1M = 4.15g =
0.1N
1liter = 0.1M 16.6g = 0.1N

Potassium iodate
KIO3, FW = 214 Eq. = 35.73g/l
250ml 0.0167M = 0.892g = 0.1N

Iodine, I2
FW = 253.18, Eq. =
127g/l
1 liter 0.05M = 12.7g =
0.1N
(use 13g Iodine and add
25g KI

Copper sulphate
CuSO4.5H2O, FW = 249.68, Eq. = 249.68g/l
250ml 0.1M = 6.24g = 0.1N
Note: add anhy. NaCO3 untill a slight permanent bluish colour
is formed. Add a little acetic acid to get a clear blue colour,
then make up to the mark.

Copper sulphate,
anhydrous
CuSO4, FW = 159.6, Eq.
= 159.6g/l
250ml 0.1M = 4g =0.1N

Bleaching powder
CaOCl2, FW = 145, Eq. = 72,5g/l
250ml 0.05M = 1.81g = 0,1N

Sodium sulphite
Na2SO3.5H2O, FW =
126.04, Eq. = 63g/l

250ml 0.05M = 1.57g =

0.1N

(use 2.5g)

TITRATIONS WITH SILVER NITRATE

1. AgNO3 + NaCl = AgCl = NaNO3 and K2CrO4 + 2AgNO3 = Ag2CrO4 + 2KNO3

2. AgNO3 + KSCN = AgSCN + KNO3 and AgSCN + Fe3+ indicator = [FeSCN]+2

Silver nitrate
AgNO3, FW = 170, Eq. =170g/l
250ml 0.1M = 4.25g = 0.1N
Sodium chloride
NaCl, FW = 58.5, Eq. = 58.5g/l
250ml 0.1M = 1.4625g = 0.1N
Ammonium thiocyanate
NH4CNS, FW = 76.12, Eq. = 76.12g/l
250ml 0.1M = 1.9g = 0.1N
1 liter 0.1M = 7.6g = 0.1N

Potassium chromate
K2CrO4, FW = 194.20, Eq. =32.4g/l
250ml 0.0167M = 0.81g = 0.1N
1 liter 0.0167M = 3.24g = 0.1N
(add a few mls dil. H2SO4 to clear)

Potassium thiocyanate
KCNS, FW = 97.18, Eq. = 97g/l
250ml 0.1M = 2.4g = 0.1N (use 3g)
1 liter 0.1M = 9.74g = 0.1N (use 12g)

REJUVINATING DIRTY HPLC COLUMNS

To Regenerate contaminated HPLC Columns

SYMPTOMS OF DIRTY COLUMNS

1. High Back Pressure.
2. Changing Retention Times.
3. Broad Peaks and Tailing.
4. Loss of Column Resolution.
In such cases, chromatographic performance will deteriorate as more and more
non-eluting compounds build up on the column stationary phase.

HPLC COLUMN REGENERATION

First try purging the column with pure acetonitrile. If this does not work, follow the
procedure below.

REGENERATION PROCEDURE FOR REVERSED-PHASE COLUMNS

1. Disconnect the column and reconnect it to the chromatograph with the flow
through the column in the reversed direction.
2. Flush out any salts/buffers with HPLC grade water. Pump 25 mL of water through
the column at 1 mL/min.
3. Flush column with 25 mL of isopropanol.
4. Flush column with 25 mL of methylene chloride.
5. Flush column with 25 mL of hexane.
6. Flush column again with 25 mL of methylene chloride.
7. Flush column again with 25 mL of isopropanol.
8. Reconnect the column to the chromatograph with the now in the proper direction.
Flush the column with the mobile phase without the buffer, then re-introduce the
buffer.
9. Equilibrate the column with 25 to 50 mL of mobile phase.
10. Inject a standard or a sample to see if performance is restored.
Note:
For some retained compounds that have fouled the column, dimethylformamide may be a better "cleaning" solvent than methylene chloride and
hexane.

REGENERATION PROCEDURE FOR NORMAL-PHASE COLUMNS

1. Connect the column to the chromatograph with the flow in the reversed direction.
2. Flush column with 50 mL of 50:50 methanol:chloroform.
3. Flush column with 50 mL of ethyl acecate.
4. Reconnect the column in the proper flow direction.
5. Equilibrate the column with 50 mL of mobile phase.
6. Inject a standard or a sample and evaluate performance.
REFERENCE:

1. MAC-MOD Analytical Inc.

HPLC TROUBLESHOOTING GUIDE

COLUMN LEAKS, RETENTION TIME, BACK

PRESSURE, PEAKS AND BASELINE PROBLEMS
START UP - PRELIMINARY CHECKS
Problem

Possible cause
Detector off

Solution
Check detector

Broken
connections to
Check connections
recorder
No peaks or
very small
No sample/Wrong Check sample. Be sure it is not
peaks
sample
deteriorated. Check for bubbles in the vials
Wrong settings on
recorder or
Check attenuation. Check gain
detector
Pump off

Start Pump

Flow interrupted

Check reservoirs. Check position of the

inlet tubing. Check loop for obstruction or
air. Check degasing of mobile phase.
Check compatibility of the mobile phase
components.

Leak

Check fittings. Check pump for leaks and

precipitates. Check pump seals.

Air trapped in the

system

Disconnect column and prime pump. Flush

system with 100% methanol or
isopropanol. Contact servicing if
necessary.

No Flow

COLUMN AND FITTINGS LEAKS

Problem

Possible cause

Column end
leaks

Loose fitting
White powder at
loose fitting

Leak at

Detector-seal

Solution
Tighten or replace fitting
Cut tubing and replace ferrule;
disassemble fitting, rinse and
reassemble.