Skip navigation.
Mexico State University


Atomic Absorption Protocols

Flame Atomic absorption instruments always use a nebulizer and a slot burner to increase the path length for sample absorption. Various fuels and oxidants are used to form atoms capable of being analyzed by atomic absorption spectroscopy. The following table lists temperatures reached with various gases used to burn aqueous sample containing metals to be analyzed.


Temperature of Some Atomic Absorption Flames



Temperature, K






2100 -2400





 Nitrous Oxide


For some elements that form refractory oxides (molecules hard to break down in the flame) nitrous oxide (N2O) needs to be used instead of air (78% N2 + 21% O2) for the oxidant. In that case, a slightly different burner head with a shorter burner slot length is used.

NOTE: The following table is for flame AAS and is based on instrument-specific conditions. In some instances, the recommended standard addition is lower than cited in this table. Dilution may be modified to meet maximum allowable limits.

Element Wave Length, (nm) Sens. Check a (mg/L)  Linear Range b (mg/L) Min.c (mg/L) Max. d (mg/25 mL) Expected Absorbance Units
Antimony  217.6   15.0 100.0 - 1.5  0.038   2.50   0.10-0.20
Barium 553.6  10.0 50.0 - 1.0  0.10  0.20  0.06-0.12
Bismuth  223.1  10.0  50.0- 1.0  0.025  1.25  0.12-0.24
Cadmium  228.8  0.5  3.0 - 0.05  0.075  0.0013  0.18-0.36
Calcium  422.7  0.5  3.0 - 0.05  0.0013  0.075  0.25-0.50
Cobalt  240.7  2.5  15.0 - 0.25  0.0063  0.375  0.08-0.16
Copper  324.8  1.5  10.0 - 0.15  0.0038  0.250 0.11-0.22 
Iron  248.3  2.5  15.0 - 0.25  0.0063  0.375 0.20-0.40
Lithium  670.8  1.0  5.0 - 0.10  0.0025  0.125  0.11-0.22
Magnesium  285.2  0.15  1.0 - 0.015  0.0004  0.025  0.25-0.50
Manganese  279.5  1.0  5.0 - 0.10  0.0025  0.125  0.22-0.44
Molybdenum  313.3  15.0  100 - 1.50  0.038   2.50  0.13-0.26
Nickel  232.0   4.0  0.40 - 0.01  0.50  20.0  0.18-0.36
Potassium  766.5   0.4  2.0 - 0.04  0.001  0.050  0.20-0.40
Silver  328.1  1.5  10.0 - 0.15  0.0038  0.250  0.11-0.22
Sodium  589.0  0.15  1.0 - 0.015  0.0004  0.025  0.25-0.50
Strontium  460.7  2.0  10.0 - 0.20  0.005  0.250  0.35-0.70
 Zinc  213.9  0.4  2.0 - 0.04  0.001  0.050  0.20-0.40

a Sensitivity Check is the concentration giving approximately 0.2 Absorbance Units (AU).

b Linear Range is the upper concentration of linear range.

c Minimum mg/L is the concentration giving 0.02 AU (Sens. Check divided by 10)

d Maximum is the upper limit in mg per 25 mL (linear range divided by 40).


Flame Atomic Absorption Spectroscopy

Flame-AAS is known as a technique with few problems related to interference effects. The interferences that occur are well defined, as are the means of dealing with them. For analysis of a few elements the type and temperature of the flame are critical; with improper conditions ionization and chemical interferences may occur.



Rich FlameIonization of the analyte atoms in the flame depletes the levels of free ground state atoms available for light absorption. This will reduce the atomic absorption at the resonance wavelength and lead to erroneous results. The degree of ionization of a metal is strongly influenced by the presence of other ionisable metals in the flame. By addition of an excess of a very easily ionized element to the blanks, standards and samples the effect of ionization can usually be eliminated. Ionization is most common in hot flames such as nitrous oxide- acetylene flames. In an acetylene-air flame ionization is most often limited to be a problem in analysis of the alkali- and alkaline-earth metals. The fuel rich flame shown on the left is not normally used for flame analysis of most metals; this yellow flame is a lower temperature than the blue flame commonly used. This photo of the AAnalyst 800 was taken with out the safety shield between the operator and the flame.


Chemical Interference

The most common type of chemical interference occurs when the sample contains components that forms thermally stable compounds with the analyte and thus reduce the rate at which it is atomized. Adding an excess of a compound that form thermally stable compounds with the interfering element eliminates chemical interference. For example, calcium phosphate does not dissociate completely in the flame. Addition of Lanthanum will tie up the phosphate allowing calcium to be atomized. A second approach to avoid chemical interference is, if possible, to use a hotter flame. Using the method of standard addition can also control chemical interference.


Physical Interference

If the physical properties as viscosity and surface tension vary considerably between samples and standards, the sample uptake rate or nebulization efficiency may be different and lead to erroneous results. Dilution of samples or method of standard addition or both can be used to control these types of interferences.


Background absorption and light scattering

Matrix components that are not 100% atomized and that has broadband absorption spectra may absorb at the analytical wavelength. Tiny solid particles in the flame may lead to scattering of the light over a wide wavelength region. The background absorption can be accounted for by using background correction techniques.



Atomic absorption spectrophotometer single- or double-beam instrument having a grating monochromator, photomultiplier detector, adjustable slits, equipped with a air-acetylene burner head and a suitable recorder or Computer. The wavelength range must be 190-800 nm.

* Hollow cathode lamp for high sensitive elements such as copper or zinc.
* Electrodeless discharge lamp for Zn may be used if available.
* Pressure reducing regulators for acetylene and air.
* Pipettes (µl) with disposable tip in various sizes.

BlueLineReagents and standards

All reagents must be of analytical grade or better.

Distilled de-ionized water
Nitric acid (HNO3)
Zinc metal, 99.999% pure (Zn)
Acetylene gas (99.99%)
Air supply

Standard stock solutions (1000 mg mL-1 or PPM (W/V))
Zn 1000 mg mL-1: Transfer 1.0000 g high purity zinc metal to a beaker. Dissolve the metal in 10 ml 1:1 HNO3. Transfer the solution to a 1000 mL volumetric flask and dilute to the mark with distilled de-ionized water. Store the solution in a labeled polyethylene bottle. Commercially available standard solutions may also be used.


Calibration standards
Calibration standards are prepared by single or multiple dilutions of the stock metal solution. Prepare a reagent blank and at least 3 calibration standards in graduated amount in the appropriate range of the linear part of the curve. The calibration standards must contain the same acid concentration as will result in the samples following processing. For precipitation samples, that would be 1% (v/v) HNO3 and for suspended particulate matter10% (v/v) HNO3. The calibration standard should be transferred to polyethylene bottles.

Instrumental general procedure

The operating procedure will vary between instrument brands, so the instrument manual should be followed carefully. The position of observation and the fuel:oxidant ratio must be optimized. Some general guidelines are outlined below

1. Light the hollow cathode lamp or electrode discharge lamp and D2-lamp if such background correction is used. Set the lamp current to the value specified by the manufacturer.
2. Position the monochromator at wavelength 213.9 nm.
3. Carefully balance the intensity of the hollow cathode lamp and the D2-lamp if such background correction is used.
4. Align the burner head to assure that the center of the light beam passes over the burner slot.
5. Light the flame and regulate the flow of fuel and oxidant to produce an oxidizing flame (lean blue).
6. Aspirate calibration blank and establish a zero point.
7. Aspirate standard solutions and construct a calibration curve.
8. Aspirate distilled water after each standard or sample.

BlueLineInstrument performance

The “characteristic concentration” (sometimes called sensitivity) is defined as the concentration of an element (mg L -1) that will absorb 1 % of the incoming radiation. This equals a signal of 0.0044 absorbance units (AU). The “characteristic concentration” is instrument dependent and is calculated as follows:

Concentration, C = (S * 0.0044 AU) / measured absorbance
C: (mg L -1)
S: Concentration of measured standard (mg L -1)

Knowing the “concentration” allows the analyst to check if the instrument is correctly optimized and performing up to specifications.

Sequence of analysis

1. Aspirate calibration blank and establish a blank level
2. Aspirate calibration blank and standard solutions and construct a calibration curve. Use at least 3 standard solutions in addition to the calibration blank to cover the linear range. Every point at the calibration curve should, if possible, be based on replicate analysis. Distilled water should be aspirated after each standard and sample.
3. A quality control standard should be analyzed to verify the calibration.
4. A calibration blank should be analyzed to check for memory effects.
5. Aspirate unknown samples.
6. Aspirate a quality control standard for every 10th sample to check for drift.
7. Samples that are found to have concentration higher than the highest standard should be diluted and reanalyzed.


Graphite Furnace General Instrument Protocol

Graphite FurnaceThe operating protocols vary between various brands and models of instruments, making it virtually impossible to give precise details of a proposed AAS method that is guaranteed to reduce interference effects. The instrument manual should be confirmed in regards of operating instructions. A careful interference study and calibration procedure as given in the particular instrument manual must be carried out by the analyst. Some general guidelines are given below.

* Allow the light source(s) a stabilization time of 10-15 minutes before analyzing.
* Set the monochromator to the appropriate wavelength.
* Align the furnace for maximum transmission of radiation.
* Carefully balance the intensity of the hollow cathode lamp and background correction.
* A temperature calibration of the furnace should be done.
* Optimize the injection position of the auto sampler capillary in such a way that the sample droplet is gently placed in the bottom of the graphite tube. A convenient sample volume for most analyses is 20 µL.
* Make sure that the silica windows in the furnace compartment are clean to ensure maximum transmission of radiation.
* All new graphite tubes must be thermally conditioned.
* For quantification of absorption signals peak area is recommended. Be certain the flame/furnace mirror is in the proper position.


Specific Details

1. The Drying step: A quick ramp (5 s) to 15oC below the boiling point of the solvent. Then a slow ramp (25 s) to reach a temperature just above the solvents boiling point. This provides a gentle evaporation without sputtering. Hold the furnace at the selected temperature until drying is complete (5- 10 s). The drying time will vary with sample volume and salt content. A purge gas flow of 250-300 ml min-1 is normally used.

2. Pyrolysis step: A pyrolysis curve should be made to find the appropriate temperature to use in this step without losing any analyte. In a pyrolysis step a typical ramp will vary between 20-50 oC s-1. Too steep ramp may cause sputtering. A purge gas flow of 250-300 ml min-1 is normally used.

3. Atomization step: An atomization curve should be made to find the appropriate temperature to use in this step. The lowest temperature that still gives maximum signal should be used in order to extend the lifetime of the graphite tube. Zero ramp time is used in this step. Gas stop during atomization is recommended.

4. Cleaning step: A tube cleaning cycle after the analyte measurement should be done to remove any remains of sample and thereby avoid memory effects. A purge gas flow of 250-300 ml min-1 is normally used.


Assessing Instrument Performance

The characteristic mass (sometimes called sensitivity) is defined as the absolute mass of an element that will absorb 1% of the incoming radiation. This equals a signal of 0.0044 absorbance units (AU). The characteristic mass may be used as an indicator of instrument optimization. Values of the characteristic masses are most often given in the instrument documentation. Experimental values for comparison can be determined by measuring the absorbance signal (area) of a known mass of analyte and calculate using the following formula:

mass = Vs * Cs*0.0044 AU / observed peak area

mass: Characteristic mass (ng)
Vs : Standard volume injected (ml)
Cs : Standard concentration (ng ml-1)

BlueLineChemical modifiers

In order to achieve better separation between analyte and matrix prior to atomization, a chemical modifier can be used. The role of the modifier is most often to stabilize the analyte making higher temperatures in the pyrolysis step possible without any loss of analyte. The concentration level of most modifier mixtures is usually in the ppm level. The injection volume most often is in the 5-20 µl region. The modifier mixture should be injected and dried prior to sample injection.

BlueLineSequence of analysis

1. Start the analysis with an “empty tube” run. If a significant signal is obtained, a cleaning step (2650 oC, 2-3 s) should be run repetitively to remove the remains in the tube. If this is not sufficient, the graphite tube should be replaced.
2. The chemical modifier solution (if used) should be checked for contamination in a separate run.
3. The blank solution should be analyzed to establish a blank level.
4. In addition to the blank standard, at least 3 standards should be selected to cover the linear range. Repeat the analysis until good agreement between replicates and a linear calibration curve is obtained.
5. A quality control standard should be analyzed to verify the calibration.
6. Samples that are found to have concentration higher than the highest standard should be diluted into range and reanalyzed.
7. To monitor the performance of the graphite tube, a mid-level standard and a blank standard should be run after every 10th sample.

Return to Instrumentation or Teaching Instruments