Sample Preparation and Standard

Operating Procedure in AAS

By
Dr. Mohammad Moniruzzaman
Principal Scientific Officer
Soil and Environment Research Division
 Sample Types

 Solid: Soil, rock, solid waste, plant materials, food

products, electronics, medicine, fertilizer, natural
products etc.
 Liquid: Drinking water, Irrigation water, surface water,
effluents, oils, petroleum.
 Gas: Gases, Airborne particles, Dust, PM 10, PM 2.5
 Sample Types

 Organic: Plant materials, food products, herbal

products, natural products, oils, petroleum etc.
 Inorganic: water, fertilizer, electronics, effluents,
medicine.
 Mixed: Solid waste, effluents, medicine, etc.
 Sample Types

 Homogenous sample: Water, oil, solid metal, fertilizer,

medicine, chemical compounds etc.
 Heterogeneous sample: Plant materials, food
products, medicine, natural products, petroleum,
electronics, effluents, Solid waste etc.
Sampling, Sample preparation and Storage
• Frequently sampling errors are commonly much greater
than analytical errors ( Reed and Rigney, 1947).
• After the gross sample has been collected, care must be
taken to avoid contamination, and to prevent the
occurrence of further chemical reactions.
• The sample must be preserved as much as possible in its
original condition, and it is necessary to maintain the
properties and the identity of the sample at all stages of
sample preparation.
• Air drying is the most accepted procedure of solid sample
preservation.
 Grinding (Homogenious)
 Grinding
• Through mixing requires that the sample be crushed and ground to
particles of uniform size.
• The purpose of grinding is to reduce heterogeneity and to provide
maximum surface area for physical and chemical reactions.

The choice of equipment depends on three factors:

• The amount of sample to be crushed or ground
• The degree of fineness to be attained
• The contamination that can be tolerated

The types of grinders used are plate grinders, ball mills, rod mills, agate
mortars, and boron carbide mortars.
•
 Sieving

Sieving:

• Sieving is an essential part of homogenizing the sample after the

grinding operation.

• The bulk sample is then screened, and the rejected material ground
again, and rescreened until the whole sample passes through the
sieve.

• The fraction <0.5 mm, preferred in modern microanalysis

provides the needed margin of safety.
• It is also believed that the soil fraction <2 mm represents more
accurately the total soil volume then do the finer sieve size fractions.
 Errors in grinding and Sieving

Contamination by grinding
• During the process of crushing and grinding, some contamination
from the apparatus occurs due to abrasion with the sample.

• Crushing generally results in less abrasion of the apparatus than

does grinding.

• Hardened steel grinders are very susceptible to abrasion, even by

soil minerals that are less hard than quartz.

• Agate mortars have been found more suitable for this purpose
 Errors in grinding and Sieving

Contamination in Sieving
• Brass and copper screens are usually employed in sieving
because of their resistance to corrosion and rusting.
• However, some abrasion of the brass or Cu during
sieving may take place, causing the sample to become
contaminated with metal.
• Use dry sample to reduce chemical reaction.
• However in an analysis of trace elements, their use
would be the reasons for some errors. In such an
analysis, silk bolting cloth may be used for screening.
Sample Preparation for AAS
 Analysis of Plant, Soil, Food Stuff Materials

1. Digestion of Sample – Ashing

2. Avoidance of Contamination
3. Minimization of Sample Loss, especially by
Volatilization
 Ashing
Removing of organic constituents by volatilization.

Definition of Ashing

• Removal of organic Matrix

• By converting into gaseous components
• Subsequent volatilization
• Inorganic residue for analysis of elements
 Types of Ashing
Dry ashing
Wet ashing (digestion)

Requirements for ashing in Trace Elements Analysis

• Complete removal of all organic components of

the sample
• Quantitative retention of all elements to be
determined within the inorganic residue.
• Avoiding contamination from environment or
otherwise with trace elements to be determined.
 Dry ashing

Digestion Procedure

Dry Ashing:
 Samples of biological origin should be dried at about 80 ° C
for about 12 hours.

 Ashing is done at about 450-500 ° C until which ash is

obtained. Muffle furnace is used to destroy the organic
portion of the sample. Dissolve it in mineral acids.

 Mg (NO3)2 is commonly used as an ashing aid.

Wet digestion
 Oxidizing mineral acids
 HNO3, HNO3: HCl (1:3), HClO4, HNO3/ H2SO4
 HNO3/ HClO4,- HNO3/ H2SO4/ HClO4
 Hydrogen peroxide
 30% H2O2, 50% H2O2/ H2SO4
 HNO3/ H3PO4/ H2O2
 HNO3/ H2O2
 H2O2/ Fe2+
 Oxidizing alkaline solution
 KMnO4 in KOH
 NaOCl in NaOH
Common procedure for wet digestion

 Make the sample oven dry (moisture free)

 Digest the samples with HNO3/ HClO4 or Aqua regia in a hot
plate

 Nitric Acid is most commonly used acid because

 Its chemical compatibility

 Oxidizing ability
 Purity and low cost
 All nitrate salts are soluble in water
 Boiling point of HNO3 is 83oC
 Wet Ashing or Digestion

Plant:
 Finely grind the plant tissue samples after drying at
approximately 80°C for 12 hours.
 Weigh 1.00± 0.05 g of plant tissue into a 100 mL beaker
 Add 10 mL HNO3 and 5 mL HClO4 cover with a watch
glass, and digest with heating to a final volume 3 to 5 mL
(Taking the solution to dryness can cause loss of the
more volatile elements e.g. As, Se, etc.)
 Wet Ashing or Digestion
 Add 10 to 15 mL of deionized/ distilled water and filter the
digest solution through a filter paper ( Whatman No. 42/44 or
any other good quality filter paper)
 Wash the beaker with 10 mL water and pass the water
through filter paper
 Finely wash the filter paper with water and dilute the filtrate
to volume with water.
 Note

Because of potential contamination from the

regents used it is a must to use a reagent
blank prepared using the procedure described
above but excluding the tissue sample.
 Caution

Always add nitric acid to the tissue samples ( or any samples

having more than 5% organic matter) before adding perchloric
acid. Perchloric acid can react explosively with unreacted
organic materials. Because all organic material is destroyed
before allowing the nitric acid to completely evaporated
( Robert A. Isaac and Jack D. Kerber, 1971)
 Fusion of Soil Sample or Rock sample
 Fusion with Sodium Carbonate is mainly used

 Lithium borate, Potassium pyrosulphate or

potassium pyrophosphate may also be used
 Fusion of Soil Sample

• Sodium carbonate fused sample is used for the

determination of Si, Al, Fe, Ti, Ca, Mg, Mn and other
metals.
• The fusion of silicates with anhydrous Na2CO3 brings them
into forms that are completely soluble in HCl.
• A large excess of the Na2CO3 flux must be used to ensure
the complete dissolution of the fused sample in acid.
• The large Na2CO3 /sample ratio is also desirable so that
both the temperature and time of fusion can be kept to a
minimum.
• Na2CO3/ sample ratio is 4:1 by weight.
• Temperature 900-10000C in a muffle furnace.
 Fusion of Soil Sample

• Several possible digestion methods can be used to

determine the total quantity of Ni, Cu, Zn, and Cd in
soil samples.

• Fusion of soil samples with lithium metaborate

according to the procedure of Medlen et al. (1969) can
be used to prepare samples for Ni, Cu and Zn analysis.

• These methods cannot be used to prepare samples for

Cd, As, Se determinations because of the loss of Cd,
As, Se at high fusion temperatures.
 Digestion with HF, H2SO4 and HClO4
 The use of HF for silicate decomposition avoids the
introduction of appreciable quantities of extraneous ions,
particularly the alkali ions, thereby making it very desirable
for the determination of alkali elements.
 It is also used to determine Mg, Ca, Fe, Ti, and Al ( Jackson,
1958; Bennett and Hawley, 1965)
 Treatment with HNO3 to oxidize the organic matter in soils
that are high in organic matter before H2SO4 and HClO4 are
obviates the danger of explosion by reaction with HClO4.
 The HClO4 is sometimes preferred to H2SO4 as the residual
acid because it produces less interferences in spectrometric
determination ( Pratt, 1965).
 Digestion with HF
• An advantageous single HF- decomposition
atomic absorption technique has been
successfully developed for 50 to 100 mg
samples of rocks. Sediments and soils by use
of a closed vessel and H3BO3 to conserve Si, as
Silicon is lost in the form of SiF4.
 Digestion with HF
• A period of about 2 hours at 25 to 1100C is
usually sufficient to dissolve the silicates.
• A higher temperature may be required for
some refractory compounds up to 1700C in a
Teflon vessel.
• However, a larger period of digestion time is
more effective than heating at a higher
temperature.
Preparation of Standard
 Preparation of Standard
 Primary Standard Compounds (Pure Salts, Metals
etc.)
 Secondary Standard Compounds (All Acid, Base,
hygroscopic compounds)
 A stock should be a solution of a simple salt of one
metal. The salt should of analytical reagent grade and
must be highly soluble in solvent.
 Pure metals themselves are used where possible,
particularly if their salts are not stable enough on the
shelf to be considered as primary solution standard
 CRM (certified reference material)
 Preparation of Standard

The general technique is to prepare relatively concentrated stock

solution for each element
(Primary stock solution) CRM
dilution

Secondary Stock Solution

Or Working solution
dilution

Working standard

Arsenic standard solution:

Stock Solution of 500 µg of As /mL : Dissolve 0.660 g of arsenic trioxide
(As2O3) in 25 ml of 10% NaOH diluted to 1 liter with water and mix. Store
in Plastic container.
 Preparation of Standard

 For the sake of convenience it is worthwhile to

weigh out the exact quantity of the salt or metal to
give a stock solution of 1000 µg mL -1 / mg L-1 / ppm
of the element in question in the form of ion.
 Most of the stock solution prepared as directed
may be stored in polythene bottles for six months
or more.
 Very dilute standards, 1 ppm and less should not
be used for more than one or two days.
 Reagent Solution
 Reagent solutions are those which are added to
standards and samples to overcome interferences or
simply to the standard in order to match the
samples.
 They are invariably used in concentrated form and
therefore, they must be a high degree of purity,
particularly with respect to the element being
determined.
 Lanthanum chloride is used for calcium
determination.
 When the composition of a standard has to be
matched with that of a sample the matrix element to
be added must be in a high state of purity.
• Thank you..