Atomic Absorption Analysis Cookbook

Section 10

Analysis of Medicines
Analysis of Biological Substances
Atomic Absorption Analysis Cookbook Section 10
Table of Contents

Introduction
18. Analysis of Medicines
18.1 Purity Testing
18.1.1 Furnace Atomic Absorption Method
18.1.2 Flame Atomic Absorption Method
18.1.3 Reduction Vaporization Atomic Absorption Method
18.2 Quantitation
18.2.1 Flame Atomic Absorption Method
18.3 Test Method for Rubber Stoppers used for Transported Liquids
18.3.1 Sample Pretreatment
18.3.2 Flame Atomic Absorption Method

19. Analysis of Biological Substances

19.1 Blood Serum Analysis Method
19.1.1 Furnace Atomic Absorption Method
19.1.2 Flame Atomic Absorption Method
19.2 Whole Blood Analysis Method
19.2.1 Furnace Atomic Absorption Method
19.3 Urine Analysis Method
19.3.1 Furnace Atomic Absorption Method
19.4 Tissue Analysis Method
19.4.1 Sample Pretreatment
19.4.2 Furnace Atomic Absorption Method
19.4.3 Flame Atomic Absorption Method

1
Introduction
Cookbook Section 10 describes the analysis methods for medicines and biological substances.
With respect to the analysis methods for medicines, the Japanese Pharmacopoeia Revision 13
specifies the elements that can be analyzed using the atomic absorption method. Using these
described analysis methods as a standard, the techniques were modified in the best way for analysis
using the Shimadzu Atomic Absorption Spectrophotometer.
The analysis methods for biological substances are based on the tests used by the Applications
Technology Division of the Kyoto Customer Support Center. Note that since there is a great
variation among the biological substances depending on the specimen composition, the described
pretreatment methods, interference during measurement, background absorption, flame conditions,
etc. may not be the most optimum for the application.
Because the measurement conditions are set with respect to the Shimadzu AA-6000 Series Atomic
Absorption Spectrophotometer, it is necessary to modify the measurement conditions when using
another type of atomic absorption spectrophotometer so that the calibration curve concentration
ranges match.

2
18 Analysis of Medicines
18.1 Purity Testing
Reference Materials
Japanese Pharmacopoeia Revision 13, Japanese Pharmacopoeia Commentary Editorial
Committee, Hirokawa Book Store
18.1.1 Furnace Atomic Absorption Method
a) Target Elements
Pb
b) Sample Pretreatment and Measurement Procedure
• Pb (matrix: refined white sugar)
Reagents
1) Pb standard solution (0.02µg Pb/mL): Refer to Cookbook Section 2,
Section 3 Preparing Standards
2) Palladium (II) nitrate solution (100µg Pd/mL): Add 15mL of nitric
acid (1+1) to 0.108g of palladium (II) nitrate, heat to dissolve, and
bring volume to 500mL using water.
3) Nitric acid: For measurement of toxic metals

Pretreatment
Accurately weigh out 0.050g of sample, transfer to a poly
tetrafluoroethylene decomposition container, add 0.5mL of nitric acid to
dissolve, seal with a cap, and heat at 150°C for 5 hours. After cooling,
accurately add enough water to bring the total volume to 5.0mL. This will
be used as the sample solution.

Procedure
Transfer precisely 1.0mL of the pretreated sample solution to each of four
2mL micro volumetric flasks. Accurately add the Pb standard solution
(0.02µg Pb/mL) in incrementally increasing volumes from 0.1 - 0.6mL to
three of the four flasks, leaving the fourth flask empty. Then add 0.2mL of
palladium (II) nitrate solution (100µg Pd/mL) to each of the four flasks,
and bring up to volume with water. Use these solutions for measurement.

3
 Measurement
Measurement wavelength 283.3nm
Calibration curve concentration range 2 - 15µg/mL (Standard
Addition Method)
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 2)
Tube High-density graphite tube
Sample injection volume 20µL
Heating conditions

TEMP (°C) TIME (sec) HEAT GAS (L/min)

STAGE 1 120 15 R 0.1
2 250 10 R 0.1
3 600 10 R 1.0
4 600 10 S 0.0H
5 600 3 S 0.0H
6 2000 3 S 0.0
7 2500 2 S 1.0

Assessment: ≤ Pb 0.5ppm

18.1..2 Flame Atomic Absorption Method

a) Target Elements
Cd, Cu, Na, Pb, Zn
b) Sample Pretreatment and Measurement Procedure

• Cd (matrix: ordinary water)

Reagents
1) Cd standard solution (1µg Cd/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards
2) Ammonium citrate solution (250g/L): Dissolve 25.0g of citric acid in
water, and bring volume to 100.0mL using water.
3) Bromthymol blue solution (0.1w/v%): Dissolve 0.1g of Thymol Blue
in ethyl alcohol (95%). Bring total volume of solution to 100.0mL
with ethyl alcohol.

4
 4) Ammonium sulfate solution (400g/L): Dissolve 40.0g of ammonium
sulfate in water, and bring volume to 100.0mL with water.
5) Diethyldithiocarbamic acid sodium salt solution (5w/v%): Dissolve
5.0g of diethyldithiocarbamic acid sodium salt in water, and bring
total volume to 100.0mL.
6) 4-methyl-2-pentanone (MIBK)
7) Nitric acid, aqueous ammonia

Procedure
1) Transfer 50.0mL of the sample solution to a 100mL separatory
funnel. Add 0.5mL of nitric acid, shake well to mix the solutions, and
set aside for 1 hour. To this solution, add 10.0mL of ammonium
citrate solution (250g/L) and 2 drops of bromthymol blue solution
(0.1w/v%). Add aqueous ammonia until the yellow color of the liquid
turns green. To this solution, add 10.0mL of ammonium sulfate
solution (400g/L) and 5.0mL of diethyldithiocarbamic acid sodium
salt solution (5w/v%) and mix. After setting aside for several minutes,
add 10.0mL of MIBK and shake vigorously to mix. Set aside, and
then collect the MIBK phase, using this as the sample solution.
2) For the standard solution, take 0.5mL of Cd standard solution (1µg
Cd/mL) and dilute this to 50.0mL with water. Then transfer to a
100mL separatory funnel. The rest of the preparation procedure for
the standard solution is the same as that performed for the sample.

5
Measurement
Measurement wavelength 228.8nm
Standard concentration 0.05µg/mL (concentration
after extraction)
Measurement conditions
Lamp current 8mA
Slit width 0.5nm
Lamp mode BGC-D2
Burner height 7mm
Support gas Air
Fuel gas flow rate
C2H2 0.8L/min (If the flame
appears red when spraying
sample, decrease the amount
of sample being suctioned.)

Assessment: Absorbency of sample solution at measurement is to be less than

that of standard solution (≤0.01mg/L)

! Cu (matrix: ordinary water)

Reagent
1) Cu standard solution (10µg Cu/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards
2) Nitric acid

Procedure
1) Add 0.5mL of nitric acid to 50.0mL of sample, shake solution to mix,
and set aside for 1 hour. This is used as the sample solution.
2) For the standard solution, take 5.0mL of Cu standard solution (10µg
Cu/mL), accurately add water to bring solution volume to 50.0mL,
then add 0.5mL of nitric acid.

6
 Measurement
Measurement wavelength 324.7nm
Standard solution concentration 1µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 15)

Assessment: Absorbance of sample solution at measurement is to be less than that

of standard solution (≤1mg/L)

! Na (matrix: calcium polystyrene sulfonate)

Reagents
Na standard solution (50µg Fe/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards

Pretreatment Procedure
Precisely weigh 1.0g of dried sample into a 50mL beaker, and add 5.0mL
of hydrochloric acid (3 mol/L) to disperse the sample. Prepare a 50mL
volumetric flask to serve as a receptacle, and above this, a 12mm i.d.
chromatographic column which is 70mm in length. The base of the column
should be filled with glass wool. Filter the sample solution through the
apparatus, and throroughly wash with a small amount of hydrochloric acid
(3mol/L). Further, continue the extraction using hydrochloric acid until the
solution volume is about 45mL. Add water to bring the solution to volume
(50mL).

Procedure
1) Precisely measure out a 2.0mL aliquot of the pretreated sample, and
accurately add hydrochloric acid (0.02 mol/L) to bring the total
volume to 500mL. This is used as the sample solution.
2) For the standard solutions, accurately add Na standard solution (50µg
Na/mL) in incrementally increasing volumes from 1.0 – 6.0mL to
several 100mL volumetric flasks, then bring up to volume with
hydrochloric acid (0.02 mol/L). Use these solutions for measurement.

7
Measurement
Measurement wavelength 589.0nm
Calibration curve concentration range 0.5 - 3µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 24)

Note: If the absorbance of the standard solution exceeds 0.5, adjust the
burner angle so that the absorbance of the standard solution having the
highest concentration is about 0.5.

Assessment: ≤Na 1%

! Pb I (matrix: titanium oxide)

Reagents
1) Pb standard solution (10µg Pb/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards
2) Ammonium citrate solution (450g/L): Dissolve 45.0g of citric acid in
water, and bring volume to 100.0mL using water.
3) Ammonium sulfate solution (400g/L)
4) Thymol Blue solution (0.1w/v%)
5) Dithizone + n-butyl acetate solution (2g/L): Add 1.0g of dithizone
(1.5-diphenylthiocarbazone) to 500mL of n-butyl acetate solution,
mix well to dissolve, then filter using type 5A filter paper.
6) Ammonia solution (10%): Dilute 10.0mL of aqueous ammonia to a
volume of 100.0mL.
7) Potassium hydrogensulfate: Reagent grade
Reagents 3) and 4) are prepared in the same way as reagents 2) and 3)
used for Cd analysis

Pretreatment procedure
Weigh 1.0g of sample into a platinum crucible, then add 10.0g of
potassium hydrogensulfate. Heat gently at first, then while occasionally
shaking, heat strongly until the liquid contents becomes transparent. After
cooling, add 20.0mL of ammonium citrate solution (450g/L) and 50.0mL
of water, and heat over a water bath to dissolve. After cooling, add water to
bring solution volume to 100.0mL. Use this as the sample source solution.

8
Procedure
1) Transfer 25.0mL of the sample source solution to a 100mL separatory
funnel. Add 10.0mL of ammonium sulfate solution (400g/L) and 5
drops of thymol blue solution (0.1w/v%), then accurately add 20.0mL
of Dithizone + n-butyl acetate solution (2g/L). Shake for 10 minutes
to mix. Set aside, and collect the n-butyl acetate phase to use for
analysis.
2) For the standard solution, transfer 6.0mL of Pb standard solution (10µ
g Pb/mL) to a platinum crucible. The rest of the procedure is the same
as that described for the sample.

Measurement
Measurement wavelength 283.3nm
Standard concentration 3µg/mL (concentration after
extraction)
Measurement conditions
Lamp current 10mA
Slit width 0.5nm
Lamp mode BGD2
Burner height 7mm
Support gas Air
Fuel gas flow rate C2H2 0.8L/min (If
the flame appears red when
spraying sample, decrease the
amount of sample being
suctioned.)

Assessment: Absorbance of sample solution at measurement is to be less than that

of standard solution (≤0.24mg/L)

9
 ! Pb II (matrix: ordinary water)
Reagents
1) Pb standard solution (10µg Pb/mL): Same as for Pb I reagent
2) Ammonium citrate solution (250g/L)
3) Bromthymol blue solution (0.1w/v%)
4) Ammonium sulfate solution (400g/L)
5) Diethyldithiocarbamic acid sodium salt solution (5w/v%)
6) 4-methyl-2-pentanone (MIBK)
7) Nitric acid, aqueous ammonia
Note: Reagents 2) - 7) are the same as reagents 2) - 7) for the Cd analysis

Procedure
1) Transfer 50.0mL of the sample solution to a 100mL separatory
funnel. Add 0.5mL of nitric acid, shake well to mix the solutions, and
set aside for 1 hour. The rest of the preparation procedure for the
sample solution is the same as that described in Cd Procedure 1).
2) For the standard solution, take 0.5mL of Pb standard solution (10µg
Pb/mL) and dilute this to 50.0mL with water. Transfer to a 100mL
separatory funnel. The rest of the preparation procedure for the
standard solution is the same as that performed for the sample.

Measurement
Measurement wavelength 283.3nm
Standard concentration 0.05µg/mL (concentration
after extraction)
Measurement conditions Same as for Pb I

Assessment: Absorbance of sample solution at measurement is to be less than

that of standard solution (≤1mg/L)

18.1.3 Reduction Vaporization Atomic Absorption Method

a) Target Elements
Hg
b) Sample Pretreatment and Measurement Procedure

10
! Hg I (matrix: hydrochloric acid, dilute hydrochloric acid)
Reagents
1) Hg standard solution (0.1µg Hg/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards
2) Stannous chloride solution (10w/v%): Add 60.0mL of sulfuric acid (1
part sulfuric acid, 20 parts water mixture) to 10.0g of tin (II) chloride
dihydrate. Mix while heating to dissolve. After cooling, dilute with
water to a volume of 100.0mL.

Procedure
1) For hydrochloric acid samples, accurately add water to 20.0mL of
sample to bring the volume to 100.0mL. Use this as the sample
solution.
For dilute hydrochloric acid samples, accurately add water to 80.0mL
of sample to bring the volume to 100.0mL. Use this as the sample
solution.
2) For the standard solution, use water to dilute 8.0mL of Hg standard
solution (0.1µg Hg/mL) to 100.0mL.

Measurement
Connect the MVU-1A Mercury Vaporization Unit to the atomic absorption
spectrophotometer, and measure the sample solution and the standard
solution. Refer to the MVU-1A instruction manual for operation
instructions.

Measurement wavelength 253.7nm

Standard concentration 8ng/mL
Measurement conditions
Lamp current 4mA
Slit width 0.5nm
Lamp mode BGC-D2

Assessment: Absorbance of sample solution at measurement is to be less than that

of standard solution (For hydrochloric acid, ≤0.04ppm; for dilute
hydrochloric acid, ≤0.01ppm).

11
! Hg II (matrix: sodium hydroxide)
Reagents
1) Hg standard solution (0.1µg Hg/mL)
2) Stannous chloride solution (10w/v%)
3) Potassium permanganate solution (60g/L): Dissolve 6.0g of
potassium permanganate in water, and dilute with water to 100.0mL.
4) Hydroxylamine hydrochloride solution (200g/L): Dissolve 20.0g of
hydroxylamine hydrochloride in water, and dilute with water to
100.0mL.
Note: Reagents 1) and 2) are the same as reagents 1) and 2) for the Hg I analysis.

Pretreatment Procedure
1) Dissolve 2.0g of sample and 1.0mL of potassium permanganate
solution (60g/L) in 30.0mL of water. Gradually add hydrochloric acid
(containing no Hg) to neutralize the solution, then add 5.0mL of
sulfuric acid (1+1). After adding enough hydroxylamine
hydrochloride solution (200g/L) to dissolve the manganese dioxide
precipitate, accurately add water to bring the total volume to
100.0mL. Use this as the sample solution.

Procedure
1) The pretreated sample solution is measured just as it is.
2) For the standard solution, add 1.0mL of potassium permanganate
solution (60g/L) to 2.0mL of Hg standard solution (0.1µg Hg/mL),
and the same amounts of hydrochloric acid and hydroxylamine
hydrochloride solution (200g/L) used to prepare the sample solution.
Accurately add water to bring the total volume to 100.0mL.

Measurement
Same as that for Hg I (Using standard solution concentration of 2ng/mL)
Assessment: Absorbance of sample solution at measurement is to be less than that
of standard solution (For hydrochloric acid, ≤0.04ppm; for dilute
hydrochloric acid, ≤0.1ppm).

12
! Hg III (matrix: gelatin, refined gelatin)
Reagents
Same as reagents 1) - 4) for the Hg II analysis.

Pretreatment Procedure
1) Weigh 2.0g of sample into a decomposition flask. Add 20.0mL of
sulfuric acid (1+1) and 100.0mL of potassium permanganate solution
(60g/L). Connect a circulating current cooling system, heat gently,
and boil for 2 hours. If the solution becomes clear during this time,
decrease the temperature to about 60°C, and add another 5.0mL of
potassium permanganate solution (60g/L). Boil again, and repeat this
procedure until the manganese dioxide precipitate persists for about
20 minutes. After cooling, add hydroxylamine hydrochloride solution
(200g/L) until the manganese dioxide precipitate disappears, and then
accurately add water to bring the volume to 150.0mL. Use this as the
sample solution.

Procedure
1) The pretreated sample solution is measured just as it is.
2) For the standard solution, transfer 2.0mL of Hg standard solution (0.1
µg Hg/mL) to a decomposition flask. Add 20.0mL of sulfuric acid
(1+1) and 100.0mL of potassium permanganate solution (60g/L) and
perform the same procedure as that used to prepare the sample
solution. Use this as the standard solution.

Measurement
Same as that for Hg I (Using standard solution concentration of
1.33ng/mL)

Assessment: Absorbance of sample solution at measurement is to be less than that

of standard solution (For hydrochloric acid, ≤0.04ppm; for dilute
hydrochloric acid, ≤0.1ppm).

13
18.2 Quantitation
Reference Materials
Japanese Pharmacopoeia Revision 13, Japanese Pharmacopoeia Commentary Editorial
Committee, Hirokawa Book Store

18.2.1 Flame Atomic Absorption Method

a) Target Elements
Ag, Al, Au, Bi, K, Na, Zn
b) Sample Pretreatment and Measurement Procedure

! Ag (matrix: sulfadiazine silver)

Reagents
Ag standard solution (50µg Ag/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards

Sample Pretreatment
Weigh out approximately 0.05g of dried sample. Dissolve in 2.0mL of
nitric acid, and accurately dilute with water to a volume of 100.0mL.

Procedure
1) Transfer 1.0mL of the pretreated sample solution to a 100mL
volumetric flask, add 2.0mL of nitric acid and bring up to volume
with water. Use this as the sample solution.
2) For the standard solutions, accurately transfer Ag standard solution
(50µg Ag/mL) to several 100mL volumetric flasks in incrementally
increasing volumes from 1.0 - 6.0mL. Add 2.0mL of nitric acid to
each, and bring up to volume with water. Use these solutions for
measurement.

Measurement
Measurement wavelength 328.1nm
Calibration curve concentration range 0.5 - 3µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 1)

14
 Assessment: Ag content 28.7 - 30.8%

! Al (matrix: Aldioxa)
Reagents
Al standard solution (200µg Al/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards

Sample Pretreatment
Weigh out precisely 0.2g of sample, add 50.0mL of hydrochloric acid
(1+4), and carefully heat to dissolve. After cooling, accurately add
hydrochloric acid (1+4) to a volume of 100.0mL.

Procedure
1) Transfer 5.0mL of the pretreated sample solution to a 50mL
volumetric flask, and bring up to volume with water. Use this as the
sample solution.
2) For the standard solutions, accurately transfer Al standard solution
(200µg Al/mL) to several 100mL volumetric flasks in incrementally
increasing volumes from 2.0 - 10.0mL. Add 1.0mL of hydrochloric
acid to each, and bring up to volume with water. Use these solutions
for measurement.

Measurement
Measurement wavelength 309.3nm
Calibration curve concentration range 10 - 40µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 2)

Assessment: Al content 11.1 - 13.0%

! Au (matrix: sodium aurothiomalate)

Reagents
Au standard solution (50µg Au/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards

15
 Sample Pretreatment
Accurately weigh out 0.7g of sample. Add 10.0mL of water and 20.0mL of
nitric acid (1+1). Shake well and bring volume to 100.0mL. Filter and
discard the first 20.0mL of filtrate. Carefully collect the next 10.0mL of
filtrate, and bring to a volume of precisely 100.0mL using water.

Procedure
1) Transfer 2.0mL of the pretreated sample solution to a 50mL
volumetric flask, and bring up to volume with water. Use this as the
sample solution.
2) For the standard solutions, accurately transfer Au standard solution
(50µg Al/mL) to several 50mL volumetric flasks in incrementally
increasing volumes from 2.0 - 10.0mL. Bring up to volume with
water. Use these solutions for measurement.

Measurement
Measurement wavelength 242.8nm
Calibration curve concentration range 2 - 10µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 4)

Assessment: Au content 49.0 - 52.5%

! Bi (matrix: phellodendron, albumin tannate and bismuth subnitrate powder)

Reagents
Bi standard solution (100µg Bi/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards

Sample Pretreatment
Weigh out exactly 0.7g of sample. Add 10.0mL of water and 20.0mL of
nitric acid (1+2). Shake well and add water to a volume of 100.0mL. Filter
and discard the first 20.0mL of filtrate. Carefully collect the next 10.0mL
of filtrate, and bring to a volume of precisely 100.0mL with water.

16
 Procedure
1) Transfer 10.0mL of the pretreated sample solution to a 100mL
volumetric flask. Bring up to volume with nitric acid (1+100). Use
this as the sample solution.
2) For the standard solutions, accurately transfer Bi standard solution
(100µg Bi/mL) to several 100mL volumetric flasks in incrementally
increasing volumes from 5.0 - 15.0mL. Bring up to volume with nitric
acid (1+100). Use these solutions for measurement.

Measurement
Measurement wavelength 223.1nm
Calibration curve concentration range 5 - 15µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 8)

Assessment: Bi content 12.9 - 16.3%

! K I (matrix: calcium polystyrene sulfonate)

Reagents
K standard solution (5mg K/mL): Refer to Cookbook Section 2, Paragraph
3 Preparing Standards

Sample Pretreatment
Weigh out precisely 1.0g of dried sample. Transfer to a glass container
equipped with a stopper. Add 50.0mL of K standard solution (5mg K/mL),
and shake for 2 hours to mix. Filter and discard the first 20.0mL of filtrate.
Carefully collect the next 5.0mL of filtrate, and bring to a volume of
exactly 100.0mL with hydrochloric acid (0.02mol/L).

Procedure
1) Accurately measure out 10.0mL of the pretreated sample solution,
and bring to a volume of 1000.0mL with hydrochloric acid
(0.02mol/L). Use this as the sample solution.

17
 2) For the standard solutions, dilute several aliquots of K standard
solution (5mg K/mL) using hydrochloric acid (0.02mol/L), so that the
range of final K concentrations is 0.5 - 2.5µg/mL. Use these solutions
for measurement.

Measurement
Measurement wavelength 766.5nm
Calibration curve concentration range 0.5 - 2.5µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 19)
Note: If the absorbance of the standard solution exceeds 0.5, adjust the burner
angle so that the absorbance of the standard solution having the highest
concentration is about 0.5.

Assessment
K replacement volume: K replacement amount in 1.0g of dried sample
0.053 - 0.071g
K replacement volume calculation
K replacement amount (mg) corresponding to 1.0g dried sample =
X − 100Y
W
where:
Y: K content (mg) in 1000.0mL of sample solution
X: K amount (mg) in 50.0mL of K standard solution before
replacement
W: Amount of dried sample (g) used

! K II (matrix: sodium polystyrene sulfonate)

Reagents
K standard solution (5mg K/mL): Same as for K I

18
Sample Pretreatment
Accurately weigh out 1.5g of the above formula-converted dried sample,
and transfer to a glass container equipped with a stopper. Add 100.0mL of
K standard solution (5mg K/mL), and shake for 15 minutes to mix. Filter
and discard the first 20.0mL of filtrate. Carefully collect the next 10.0mL
of filtrate, and bring to a volume of 100.0mL with water.

Procedure
1) Accurately measure out 10.0mL of the pretreated sample solution,
and bring to a volume of 1000.0mL with water. Use this as the
sample solution.
2) For the standard solutions, dilute several aliquots of K standard
solution (5mg K/mL) using water, so that the range of final K
concentrations is 1 - 5µg/mL. Use these solutions for measurement.

Measurement
Measurement wavelength 766.5nm
Calibration curve concentration range 1 - 5µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 19)
Note: If the absorbance of the standard solution exceeds 0.5, adjust the burner
angle so that the absorbance of the standard solution having the highest
concentration is about 0.5.

19
 Assessment
K replacement volume: K replacement amount in 1.0g of the
aforementioned formula-converted dried sample 0.110 - 0.135g
K replacement volume calculation
K replacement amount (mg) corresponding to 1.0g of the aforementioned
formula
X − 100Y
converted dried sample =
W
where:
Y: K content (mg) in 1000.0mL of sample solution
X: K amount (mg) in 100.0mL of K standard solution before
replacement
W: Amount of converted dried sample (g) used

! Na (matrix: sodium polystyrene sulfonate)

Reagents
Na standard solution (50µg Na/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards

Sample Pretreatment
Accurately weigh out 1.0g of of the aforementioned formula-converted
dried sample, and transfer to a glass container equipped with a stopper.
Add exactly 50.0mL of hydrochloric acid (3mol/L) and shake for 60
minutes to mix. Filter and discard the first 20.0mL of filtrate. Carefully
collect the next 5.0mL of filtrate, and bring to a volume of 100.0mL with
water.

Procedure
1) Measure out 20.0mL of the pretreated sample solution, and bring to a
volume of 1000.0mL with water. Use this as the sample solution.
2) For the standard solutions, prepare several 100mL volumetric flasks,
and transfer Na standard solution (50µg Na/mL) to these flasks in
incrementally increasing volumes from 2.0 - 6.0mL. Bring up to
volume with water. Use these solutions for measurement.

20
Measurement
Measurement wavelength 589.0nm
Calibration curve concentration range 1 - 3µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 24)
Note: If the absorbance of the standard solution exceeds 0.5, adjust the burner
angle so that the absorbance of the standard solution having the highest
concentration is about 0.5.

Assessment: Na 9.4 -11.0%

! Zn I (matrix: isophane insulin aqueous suspension)

Reagents
Zn standard solution (10µg Zn/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards

Procedure
1) According to the displayed units, accurately measure out a volume
corresponding to approximately 400 units of sample. Accurately add
1.0mL of hydrochloric acid (0.1mol/L) and 100.0mL of water. If
necessary, dilute using more water so that the Zn concentration in
1.0mL is 0.6 - 1.0µg. Use this as the sample solution.
2) For the standard solutions, transfer Zn standard solution (10µg
Zn/mL) to several 100mL volumetric flasks in incrementally
increasing volumes from 3.0 - 12.0mL. Add 1.0mL of hydrochloric
acid (0.1mol/L), and bring up to volume with water. Use these
solutions for measurement.

Measurement
Measurement wavelength 213.9nm
Calibration curve concentration range 0.3 - 1.2µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 44)
Note: If the absorbance of the standard solution exceeds 0.5, adjust the burner
angle so that the absorbance of the standard solution having the highest
concentration is about 0.5.

21
 Assessment: Zn 0.01 -0.04mg per 100 units of suspension

! Zn II (matrix: insulin)
Reagents
Zn standard solution (10µg Zn/mL): Same as for Zn I

Procedure
1) Measure out 0.01g of sample. Add precisely 1.0mL of hydrochloric
acid (0.1mol/L) and 200.0mL of water. If necessary, dilute using
more water so that the Zn concentration in 1mL is 0.6 - 1.0µg. Use
this as the sample solution.
2) For the standard solutions, perform the same procedure as for Zn I,
Procedure, 2).

Measurement: Same as for Zn I

Assessment: Zn 0.27 - 1.08%

! Zn III (matrix: insulin zinc injection aqueous susupension), crystalline insulin

zinc injection aqueous suspension), amorphous insulin zinc injection aqueous
suspension)
Reagents
Zn standard solution (10µg Na/mL): Same as for Zn I

Procedure
1) According to the displayed units, accurately measure out a volume
corresponding to approximately 200 units of sample. Add 1.0mL of
hydrochloric acid (0.1mol/L) and 200.0mL of water. If necessary,
dilute using more water so that the Zn concentration in 1mL is 0.6 -
1.0µg. Use this as the sample solution.
2) For the standard solutions, perform the same procedure as for Zn I,
Procedure, 2).

Measurement: Same as for Zn I

Assessment: Zn 0.12 -0.30mg per 100 units of suspension

22
18.3 Test Method for Rubber Stoppers used for Transported Liquids
Reference Materials
Japanese Pharmacopoeia Revision 13, Japanese Pharmacopoeia Commentary Editorial
Committee, Hirokawa Book Store

18.3.1 Sample Pretreatment

a) Impurities (Cd, Pb)
Wash the rubber stopper with water, dry it at ambient temperature, and cut it into
fine pieces. Mix the pieces well, and measure 2.0g of the pieces into a platinum
or quartz glass crucible. Add 2mL of sulfuric acid to moisten. Heat gradually to
dry, then heat at 450 - 500°C to ash. If ashing is incomplete, moisten with 1mL of
sulfuric acid, heat to dryness, and then ash. If necessary, repeat this process. After
cooling, moisten the residue with water, add 2 - 4mL of hydrochloric acid, and
heat on a water bath to evaporate to dryness. Add another 1 - 5mL of
hydrochloric acid, and heat to dissolve. Next, add 0.5 - 1mL of 50% citric acid
solution and hydrochloric acid (1+1) and 0.5 - 1mL of heated ammonium acetate
solution (40%) to dissolve the sample. (If any impurities remain, filter through a
glass filtration device.)

b) Extracted substance (Zn)

Wash the rubber stopper and dry at ambient temperature. Place this in hard glass
container. Add water at a weight 10 times that of the sample weight, and after
appropriately capping the container, place it in a high-pressure steam sterilizer
and heat at 121°C for 1 hour. Remove the glass container from the sterilizer and
set it aside until it cools to ambient temperature, then immediately remove the
rubber stopper and use the liquid as the sample source liquid.

18.3.2 Flame Atomic Absorption Method

a) Target Elements
Cd, Pb, Zn
b) Measurement Procedure
Measurement is conducted using the following procedures. For lamp current, slit
width and flame conditions, refer to Cookbook Section 3, Paragraph 6.4
Measurement Conditions According to Element.

23
! Cd
Reagents
1) Cd standard solution (1µg Cd/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards
2) Ammonium citrate solution (250g/L)
3) Bromthymol blue solution (0.1w/v%)
4) Ammonium sulfate solution (400g/L)
5) Diethyldithiocarbamic acid sodium salt solution (5w/v%)
6) 4-methyl-2-pentanone (MIBK)
Reagents 2) - 6) are the same as those described in Paragraph 18.1.2,
Cd Reagents 2) - 6)
7) Aqueous ammonia (10%): Take 10.0mL of aqueous ammonia and
dilute to 100.0mL with water.

Procedure
1) Transfer all of the pretreated sample solution to a 200mL separatory
funnel. Add 10.0mL of ammonium citrate solution (250g/L) and 2
drops of bromthymol blue solution (0.1w/v%). Add aqueous
ammonia (10%) until the yellow color of the liquid turns green. To
this solution, add 10.0mL of ammonium sulfate solution (400g/L),
and add water to bring the volume to 100.0mL. Next, add 20.0mL of
diethyldithiocarbamic acid sodium salt solution (5w/v%) and mix.
After setting aside for several minutes, add 20.0mL of MIBK and
shake vigorously to mix. Set aside, and then collect the MIBK phase,
using this as the sample solution. If necessary, filter this solution.
2) For the standard solution, transfer 10.0mL of Cd standard solution (1
µg Cd/mL) to a 200mL separatory funnel. Add 10.0ml of ammonium
citrate solution (250g/L) and 2 drops of bromthymol blue solution
(0.1w/v%). The rest of the preparation procedure for the standard
solution is the same as that performed for the sample.

24
 Measurement
Measurement wavelength 228.8nm
Standard concentration 0.5µg/mL (concentration after
extraction)

Measurement conditions: Same as that described Paragraph 16.1.2 Flame

Atomic Absorption Method, Cd Measurement Conditions

Assessment: Absorbance of sample solution at measurement is to be less than that

of standard solution (≤5ppm)
! Pb
Reagents
1) Pb standard solution (10µg Pb/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards
2) Ammonium citrate solution (250g/L)
3) Bromthymol blue solution (0.1w/v%)
4) Ammonium sulfate solution (400g/L)
5) Diethyldithiocarbamic acid sodium salt solution (5w/v%)
6) 4-methyl-2-pentanone (MIBK)
Reagents 2) - 6) are the same as those described in Paragraph 18.1.2,
Cd Reagents 2) - 6)
7) Aqueous ammonia (10%): Same as for Cd Reagents, 7)

Procedure
1) Same as Cd Procedure 1)
2) For the standard solution, transfer 1.0mL of Pb standard solution (10µ
g Cd/mL) to a 200mL separatory funnel. Add 10.0ml of ammonium
citrate solution (250g/L) and 2 drops of bromthymol blue solution
(0.1w/v%). The rest of the preparation procedure for the standard
solution is the same as that performed for the sample.

25
 Measurement
Measurement wavelength 283.3nm
Standard concentration 0.5µg/mL (concentration after
extraction)

Measurement conditions: Same as that described in Paragraph 16.1.2

Flame Atomic Absorption Method, Pb I Measurement Conditions

Assessment: Absorbance of sample solution at measurement is to be less than that

of standard solution (≤5ppm)

! Zn
Reagents
Zn standard solution (10µg Zn/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards

Procedure
1) Using 10.0mL of pretreated sample, accurately add nitric acid (1+50)
to a volume of 20mL. Use this as the sample solution.
For the blank test, perform the same procedure on water as performed
on the sample. Take 10.0mL of this solution, and accurately add nitric
acid (1+50) to a volume of 20.0mL. The resulting measurement of the
blank is used to correct subsequent standard and sample measurement
values.
2) For the standard solutions, take 1.0mL of Zn standard solution (10µg
Zn/mL), and accurately add nitric acid (1+50) to a volume of 20.0mL.

Measurement
Measurement wavelength 213.9nm
Standard concentration 0.5µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 44)

Assessment: Absorbance of sample solution at measurement is to be less than that

of standard solution (≤Zn 0.25µg/mL of eluate)

26
19 Analysis of Medicines
19.1 Blood Serum Analysis Method
19.1.1 Furnace Atomic Absorption Method
a) Target Elements
Al
Reference Materials
Shimadzu Commentary Vol. 37. No1. P75 (1980)
Shimadzu Commentary Vol. 40. No4. P17 (1983)
b) Measurement Procedure
Measurement is conducted using the following procedures. For lamp
current and slit width, refer to Cookbook Section 4, Paragraph 7.5
Measurement Conditions According to Element.

! Al
Reagents
Al standard solution (100ng Al/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards

Procedure
1) Measure 2.0mL of serum into a 10mL volumetric flask, and bring up
to volume with water. Use this solution for measurement.
2) For the standard solutions, accurately add Al standard solution (100ng
Al/mL) in incrementally increasing volumes from 0.2 - 2.0mL to
several 10mL volumetric flasks, and bring up to volume with water.
Use these solutions for measurement.

Measurement
Measurement wavelength 309.3nm
Calibration curve concentration range 2 - 20ng/mL
Tube pyrolyzed graphite tube
Sample injection volume 20µL
Heating conditions

27
 TEMP (°C) TIME (sec) HEAT GAS (L/min)
STAGE 1 120 15 R 0.1
2 250 10 R 0.1
3 800 10 R 1.0
4 800 20 S 1.0
5 800 3 S 0.0H
6 2500 3 S 0.0H
7 2700 2 S 1.0

19.1.2 Flame Atomic Absorption Method

a) Target Elements
Ca, Cu, Fe, K, Mg, Na
Reference Materials
Shimadzu Commentary Vol. 37. No1. P75 (1980)
b) Sample Pretreatment

! Ca, K, Mg, Na
Dilute the serum so that the concentration is within the calibration curve
concentration range. Use the solution for measurement. For analysis of Ca
and Mg, add La to inhibit interference.

! Cu, Fe
Transfer 2.0mL of serum to a centrifuge separation tube. Add 2.0mL of
hydrochloric acid (1+1) and 2.0mL of trichloroacetic acid solution
(200g/L) and mix well. Set aside for 5 minutes, and then centrifuge at
3000rpm for 5 minutes. Transfer the upper layer supernatant to a test tube
using a pipette, and use this solution for measurement.

c) Measurement Procedure
Measurement is conducted using the following procedures. For lamp
current, slit width and flame conditions, refer to Cookbook Section 3,
Paragraph 6.4 Measurement Conditions According to Element.

28
! Ca
Reagents
1) Ca standard solution (10µg Ca/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards
2) La solution (50g La/L): Weigh out 67.0g of lanthanum oxide
(heptahydrate) and gradually add hydrochloric acid (1+1) to dissolve.
Add water to bring the volume to 500.0mL.

Procedure
1) Measure 1.0mL of serum into a 50mL volumetric flask, add 3.0mL of La
solution (50g La/L), and bring to volume with water. Use this solution for
measurement.
For the blank test, measure 3.0mL of La solution (50g La/L) into a 50mL
volumetric flask, and bring up to volume with water. After measuring this
solution, the result is used to correct subsequent standard and sample
measurement values.
2) For the standard solutions, accurately add Ca standard solution (10µg
Ca/mL) in incrementally increasing volumes from 5.0 - 25.0mL to several
50mL volumetric flasks. Add 3.0mL of La solution (50g La/L) to each, and
bring to volume with water. Use these solutions for measurement.

Measurement
Measurement wavelength 422.7nm
Calibration curve concentration range 1 - 5µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 9)

! Cu
Reagents
1) Cu standard solution (10µg Cu/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards

29
 Procedure
1) The pretreated sample solution is used as is for measurement.
For the blank test, measure 2.0mL of water into centrifuge separation tube,
and perform the same procedure as that performed on the sample solution.
After measuring this solution, the result is used to correct subsequent
standard and sample measurement values.
2) For the standard solutions, accurately add Cu standard solution (10µg
Cu/mL) in incrementally increasing volumes from 1.0 - 5.0mL to several
50mL volumetric flasks. Add 10.0mL of hydrochloric acid (1+1) to each,
and bring up to volume with water. Use these solutions for measurement.

Measurement
Measurement wavelength 324.8nm
Calibration curve concentration range 0.2 - 1µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 15)

! Fe
Reagents
1) Fe standard solution (10µg Fe/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards

Procedure
1) Same as that for Cu 1).
2) For the standard solutions, accurately add Fe standard solution (10µg
Fe/mL) in incrementally increasing volumes from 2.0 - 10.0mL to
several 50mL volumetric flasks. Add 10.0mL of hydrochloric acid
(1+1) to each, and bring up to volume with water. Use these solutions
for measurement.

Measurement
Measurement wavelength 248.3nm
Calibration curve concentration range 0.4 - 2µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 16)

30
! K
Reagents
1) K standard solution (10µg K/mL)
2) Na standard solution (100µg Na/mL)
For 1) and 2) Refer to Cookbook Section 2, Paragraph 3 Preparing
Standards

Procedure
1) Measure 5.0mL of serum into a 100mL volumetric flask, and bring to
volume with water. Transfer a 4.0mL aliquot of this solution to
another 100mL flask and bring to volume with water. Use this
solution for measurement.
2) For the standard solutions, accurately add K standard solution (10µg
K/mL) in incrementally increasing volumes from 1.0 - 8.0mL to
several 100mL volumetric flasks. Add to each of these 6.0mL of Na
standard solution (100µg Na/mL), then bring up to volume with
water. Use these solutions for measurement.

Measurement
Measurement wavelength 766.5nm
Calibration curve concentration range 0.1 - 0.8µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 19)

! Mg
Reagents
1) Mg standard solution (5µg Mg/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards
2) La solution (50g La/L): Same as for Ca Reagents, 2)

31
 Procedure
1) Measure 0.5mL of serum into a 50mL volumetric flask. Add 3.0mL
of La solution (50g La/L) and bring to volume with water. Use this
solution for measurement.
For the blank test, measure 3.0mL of La solution (50g La/L) into a
50mL volumetric flask, then bring up to volume with water. After
measuring this solution, the result is used to correct subsequent
standard and sample measurement values.
2) For the standard solutions, accurately add Mg standard solution (5µg
Mg/mL) in incrementally increasing volumes from 1.0 - 5.0mL to
several 50mL volumetric flasks. Add 3.0mL of La solution (50g
La/L) to each, then bring up to volume with water. Use these
solutions for measurement.

Measurement
Measurement wavelength 285.2nm
Calibration curve concentration range 0.1 - 0.5µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 21)

! Na
Reagents
1) Na standard solution (10µg Na/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards

Procedure
1) Measure 1.0mL of serum into a 100mL volumetric flask, and bring to
volume with water. Transfer a 2.0mL aliquot of this solution to
another 100mL flask and bring to volume with water. Use this
solution for measurement.
2) For the standard solutions, accurately add Na standard solution (10µg
Na/mL) in incrementally increasing volumes from 1.0 - 8.0mL to
several 100mL volumetric flasks, and bring up to volume with water.
Use these solutions for measurement.

32
 Measurement
Measurement wavelength 589.0nm
Calibration curve concentration range 0.1 - 0.8µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 24)
Note: If the absorbance of the standard solution exceeds 0.5, adjust the burner
angle so that the absorbance of the standard solution having the highest
concentration is about 0.5.

19.2 Whole Blood Analysis Method

19.2.1 Furnace Atomic Absorption Method
a) Target Elements
Pb
Reference Materials
Shimadzu Commentary Vol. 40. No4. P11 (1983)

b) Measurement Procedure
Measurement is conducted using the following procedures. For lamp current
and slit width, refer to Cookbook Section 4, Paragraph 7.5 Measurement
Conditions According to Element.

! Pb
Reagents
1) Pb standard solution (50ng Pb/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards
2) Triton X solution (10w/v%): Dissolve 10.0g of Triton X-100 in water,
and dilute with water to a volume of 100.0mL.
3) Ammonium phosphate solution (10w/v%): Dissolve 10.0g of
triammonium phosphate trihydrate, and dilute with water to a volume
of 100.0mL.

Procedure
1) Measure 0.5mL of serum into a 10mL volumetric flask. Add 1.0mL
of Triton X solution (10w/v%) and 2.5mL of ammonium phosphate
solution (10w/v%), then bring up to volume with water. Use this
solution for measurement.

33
 2) For the standard solutions, accurately add Pb standard solution (50µg
Pb/mL) in incrementally increasing volumes from 0.4 - 2.0mL to
several 10mL volumetric flasks. Add 1.0mL of Triton X solution
(10w/v%) and 2.5mL of ammonium phosphate solution (10w/v%) to
each of these, then bring up to volume with water. Use these solutions
for measurement.

Measurement
Measurement wavelength 283.3nm
Calibration curve concentration range 2 - 10ng/mL
Tube High-density graphite tube
Sample injection volume 20µL
Heating conditions

TEMP (°C) TIME (sec) HEAT GAS (L/min)

STAGE 1 120 15 R 0.1
2 250 10 R 0.1
3 400 10 R 1.0
4 400 20 S 1.0
5 400 3 S 0.0H
6 1800 3 S 0.0H
7 2500 2 S 1.0

19.3 Urine Analysis Method

19.3.1 Furnace Atomic Absorption Method
a) Target Elements
Cr
Reference Materials
Shimadzu Commentary Vol. 41. No4. P61 (1984)

b) Measurement Procedure
Measurement is conducted using the following
procedures. For lamp current and slit width,
refer to Cookbook Section 4, Paragraph 7.5
Measurement Conditions According to Element.

34
! Cr
Reagents
1) Cr standard solution (25ng Cr/mL): Refer to
Cookbook Section 2, Paragraph 3 Preparing
Standards

Procedure
Transfer 10.0mL aliquots of urine to each of four
25mL volumetric flasks. Do not add any Cr standard
solution (25ng Cr/mL) to one of these flasks.
Accurately add the Cr standard solution in
incrementally increasing volumes from 1.0 - 5.0mL
to the remaining three flasks, then bring each of
these up to volume with water. Use these solutions
for measurement.

Measurement
Measurement wavelength 357.9nm
Calibration curve concentration range 1 -
5ng/mL
Tube Pyrolyzed graphite
tube
Sample injection volume 20µL
Heating conditions

TEMP (°C) TIME (sec) HEAT GAS (L/min)

STAGE 1 120 15 R 0.1
2 250 10 R 0.1
3 700 10 R 1.0
4 700 20 S 1.0
5 700 3 S 0.0H
6 2500 3 S 0.0H
7 2700 2 S 1.0

35
19.4 Tissue Analysis Method
19.4.1 Sample Pretreatment
a) Decomposition with nitric acid and perchloric acid
Weigh out 2 - 5g of the collected tissue specimen,
and transfer to a 200mL conical beaker. Add 20.0mL
of nitric acid (1+1), and heat gently to initiate
a reaction with the sample. After cooling, add
2.0mL of perchloric acid, and heat gently to
concentrate. When the contents begins to turn a
dark color, add nitric acid, 2 -3 mL at a time,
and continue heating. When the contents begin to
appear slightly yellowish or colorless, continue
heating until the white fumes of perchloric acid
are generated. Cool, then add 5.0mL of nitric acid
(1+1) and heat to dissolve the salts. After
cooling again, dilute with water to the same
volume. Use this solution for measurement.
CAUTION:
1) If during the decomposition process the sample becomes carbonized
or dried, it will explode. For this reason, be sure that nitric acid has
been added before heating.
2) If the contents should become carbonized, the target element may be
lost due to volatilization.
3) Since there is a possibility of contamination due to impurities in the
reagents or lab ware, a blank solution is prepared using reagent only,
and is processed in the same way as the sample.

b) Dry Ashing
Weigh out 2 - 10g of sample that has been air-dried, and transfer to a
100mL quartz glass beaker. Heat gently on a hot plate. Continue heating
until sufficient water has been eliminated to the point of partial
carbonization. Transfer to an electric furnace, and heat so that the
temperature rises at about 100°C per hour. Then heat at 500°C for several
hours up to 10 hours, until ashing is completed. Add 2 - 4mL of water to
the ash, and dry over a water bath. Add 5.0mL of nitric acid (1+1) to

36
 dissolve the salts, and dilute with water to a fixed volume. Use this solution
for measurement.
Note: There is a possibility of volatilization of nearly all elements. Cd will
be volatilized at an ashing temperature above 500°C. If halogens are
present, As, Sb, Sn and Zn, etc. are easily volatilized. Heating above 550°C
will cause a substantial reduction in element recovery.

19.4.2 Furnace Atomic Absorption Method

a) Target Elements
Cu, Mn

b) Measurement Procedure
Measurement is conducted using the following procedures. For lamp
current and slit width, refer to Cookbook Section 4, Paragraph 7.5
Measurement Conditions According to Element.

! Cu
Reagents
1) Cu standard solution (25ng Cu/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards

Procedure
1) The pretreated sample solution is used as is for analysis. If necessary,
dilute it with nitric acid (0.1mol/L) so that the target element
concentration is within the calibration curve concentration range.
For the blank test, prepare a blank test solution using the same
procedure as that for the sample. The resulting measurement of the
blank solution is used to correct subsequent standard and sample
measurement values.
2) For the standard solutions, accurately add Cu standard solution (25ng
Cu/mL) in incrementally increasing volumes from 1.0 - 6.0mL to
several 25mL volumetric flasks, then bring up to volume with nitric
acid (0.1mol/L). Use these solutions for measurement.

37
 Measurement
Measurement wavelength 324.8nm
Calibration curve concentration range 1 - 6ng/mL
Tube Pyrolyzed graphite tube
Sample injection volume 10µL
Heating conditions

TEMP (°C) TIME (sec) HEAT GAS (L/min)

STAGE 1 120 15 R 0.1
2 250 10 R 0.1
3 500 10 R 1.0
4 500 15 S 1.0
5 500 3 S 0.0H
6 2300 3 S 0.0H
7 2700 2 S 1.0

! Mn
Reagents
1) Mn standard solution (25ng Mn/mL): Refer to
Cookbook Section 2, Paragraph 3 Preparing
Standards

Procedure
1) The pretreated sample solution is used as is
for analysis. If necessary, dilute it with
nitric acid (0.1mol/L) so that the target
element concentration is within the
calibration curve concentration range.
For the blank test, prepare a blank test
solution using the same procedure as that for
the sample. The resulting measurement of the
blank solution is used to correct subsequent
standard and sample measurement values.

2) For the standard solutions, accurately add Mn

38
 standard solution (25ng Mn/mL) in
incrementally increasing volumes from 0.5-
3.0mL to several 25mL volumetric flasks, then
bring up to volume with nitric acid
(0.1mol/L). Use these solutions for
measurement.

Measurement
Measurement wavelength 279.5nm
Calibration curve concentration range 0.5 -
3ng/mL
Tube Pyrolyzed graphite
tube
Sample injection volume 10µL
Heating conditions
TEMP (°C) TIME (sec) HEAT GAS (L/min)
STAGE 1 120 15 R 0.1
2 250 10 R 0.1
3 600 10 R 1.0
4 600 15 S 1.0
5 600 3 S 0.0H
6 2300 3 S 0.0H
7 2700 2 S 1.0

19.4.3 Flame Atomic Absorption Method

a) Target Elements
Cd, Fe, Zn

b) Measurement Procedure
Measurement is conducted using the following procedures. For lamp current, slit
width and flame conditions, refer to Cookbook Section 3, Paragraph 6.4
Measurement Conditions According to Element.

39
! Cd
Reagents
1) Cd standard solution (10µg Cd/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards

40
 Procedure
1) The pretreated sample solution is used as is for analysis. If necessary,
dilute it with nitric acid (0.1mol/L) so that the target element
concentration is within the calibration curve concentration range.
For the blank test, prepare a blank test solution using the same
procedure as that for the sample. The resulting measurement of the
blank solution is used to correct subsequent standard and sample
measurement values.
2) For the standard solutions, accurately add Cd standard solution (10ng
Cd/mL) in incrementally increasing volumes from 1.0- 6.0mL to
several 100mL volumetric flasks, then bring up to volume with nitric
acid (0.1mol/L). Use these solutions for measurement.

Measurement
Measurement wavelength 228.8nm
Calibration curve concentration range 0.1 - 0.6µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 11)

! Fe
Reagents
1) Fe standard solution (50µg Fe/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards

Procedure
1) Same as for Cd, 1)
2) For the standard solutions, accurately add Fe standard solution (50ng
Fe/mL) in incrementally increasing volumes from 1.0- 8.0mL to
several 100mL volumetric flasks, then bring up to volume with nitric
acid (0.1mol/L). Use these solutions for measurement.

Measurement
Measurement wavelength 248.3nm
Calibration curve concentration range 0.5 - 4.0µg/mL
Measurement conditions Refer to Cookbook Section 3,

41
 Paragraph 6.4, 16)

! Zn
Reagents
1) Zn standard solution (10µg Zn/mL): Refer to Cookbook Section 2,
Paragraph 3 Preparing Standards

Procedure
1) Same as for Cd, 1)
2) For the standard solutions, accurately add Zn standard solution (10ng
Zn/mL) in incrementally increasing volumes from 1.0- 6.0mL to
several 100mL volumetric flasks, then bring up to volume with nitric
acid (0.1mol/L). Use these solutions for measurement.

Measurement
Measurement wavelength 213.9nm
Calibration curve concentration range 0.1 - 0.6µg/mL
Measurement conditions Refer to Cookbook Section 3,
Paragraph 6.4, 44)

42