SPECTROSCOPIC
CHARACTERISTICS OF HUMATES
ISOLATED FROM DIFFERET SOILTYPES
NADĚŽDA FASUROVÁ1, LUBICA POSPÍŠILOVÁ2

FACULTY OF
CHEMISTRY

1Brno University of Technology, Faculty of Chemistry, Purkyňova 118, 612 00 Brno, CzechRepublic.
2Mendel University of Agriculture and Forestry, Soil Science, Microbiology and Plant *utrition,Zemědělská 1, 613 00 Brno, Czech Republic.
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Aims of this project The aim of our work was to compare

spectroscopic properties of soil humatesisolated from different soil matrices(Haplic Cambisol, Leptic Cambisol, EutricCambisol, Haplic Chernozem). The opticalmethods (UV-VIS and SFS) in this project
were used.
 Then we compared the type of land use(arable soil x grassland) in EutricCambisol samples.

Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Fluorescence of humic substances










State transition of electrons from excited singlet state to basic level ofsinglet (light emission). Transition is permitted. (~ns)
Photoluminescence:for the excitation Xe lamp or laser are used.
Excitation and emission wavelength can be chosen and set bymonochromators. Excitation wavelength is lower than emisssion
wavelength, emission wavelength corresponds to lower energy.
Measurement of liquid or solid samples. (HK a FK in solution)
Fluorescent molecules (with conjugated double bond system, aromaticsubstances)
Humic substances (phenolic compounds, aromatic rings with aminogroups, hydroxyl groups)-mixture of these substances.
Comparing fluorescence peaks with peaks of pure substancesor with standards (IHSS)

Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Spectrofluorimeter scheme
Emission shutter
Sample cuvette

PMT
Emission monochromator
Excitation shutter

Excitation monochromator

90° angle geometryis frequently used

Xenon lamp
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Materials and methods:
Czech soil samples, 4 subtypes of Cambisols, 1 Chernozem
Type of land use: arable soil x grassland Eutric Cambisol

 Localities:

Leptic Cambisol=Ocmanice
Haplic Cambisol=Náměšť
Eutric Cambisol 1, 2=Vatín
Haplic Chernozem=Praha,
RuzyněVatínNáměšť,Ocmanice
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

www.mapy.cz

Materials and methods

Isolation of HA was made according to the IHSS method (0.5 MNaOH)
Sodium humates were prepared from HA and titration to pH=7(dialyzation MWCO 3500, lyophilization at -50°C)
Samples for UV-Vis: HS extracts were made in the mixture of 0.1
M Na4P2O7 and 0.1M NaOH
Samples for SFS: dissolving of humates in Mili-Q water, c=50mg/l
Elemental analysis (C, H, N, O contents) of isolated HA waskindly made in Engineering Test Institute Brno.
We determined total carbon content, fractional composition, andhumification degree of soil humates.

Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Materials and methods
 UV-VIS-spectrometerVarian Cary 50 Probe,

glass fiber, scan range 300 - 700 nm,(MUAF).
 SFS-Spectrofluorimeter Aminco Bowman
Series 2, scan range 200-600 nm,∆λ = (λem. - λex. ) = 55 nm, emission mode,
90° angle geometry, temperature 20 °C,
bandpass: 4 nm, voltage: 1040 V ,(BUT FCH)

Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Total organic carbon, humification
degree

TOC= was determined byshort oxidimetric titration
method by Nelson andSommers (1982).
Humification degree [HD]was calculated by using thisequation:

HA ∗100∑
HD =[%]
by Orlov (1985)
where: ΣHA=humic acidcontent, TOC=total organiccarbon content
Humic Substances in Ecosystems
8, Šoporňa 13.-17.9.2009 Slovakia

TOC

Colour and fluorescence indexes Colour index:

Q4/6= A(465)/A(665) nm,
by Orlov (1985), Podlešáková (1992)
 Fluorescence index:

F=RFI(468)/RFI(522) nm ,as a ratio of secondary and main peak

Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Table1 Fractional composition of soil samples,values of colour indexes and fluorescence indexes
Soil types

TOC
[%]

HSHA

FA

HA/FA

HD
[%]

Q4/6

F

[mg/kg][mg/kg][mg/kg]

LepticCambisol

1.32

4.14

1.44

2.7

0.4

9.0

9.1

0.64

HaplicCambisol

1.62

4.65

1.6

3.0

0.54

12.1

5.7

0.72

EutricCambisol 1arable

1.76

6.8

1.8

5.0

0.5

10.1

8.1

0.57

EutricCambisol 2grassland

2.1

7.62.9

4.7

0.6

13.8

8.3

0.94

HaplicChernozem

1.88

5.3

2.9

2.4

1.21

15.4

5.2

0.58

Humic Substances in Ecosystems
8, Šoporňa 13.-17.9.2009 Slovakia

Results
 The highest TOC content had EutricCambisol 2 (grassland) The highest HA content had Eutric Cambisol
2 and Haplic Chernozem
 The highest of HD values had Haplic
Chernozem, the lowest HD values had
Leptic Cambisol
 HS quality in grassland soil sample washigher than in arable soil (E. Cambisol)

Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Table 2 Ash free elemental analysis of soil
humic acids (in atomic %).
Sample

C[%]

H[%]

N[%]

O[%]

Ash[%]

LepticCambisol

33.45

47.44

3.07

16.05

1.7

HaplicCambisol

34.20

46.16

3.05

16.59

4.1

EutricCambisol1(arable)

32.73

46.48

2.52

18.27

6.2

Eutric35.59
Cambisol 2(grassy)

45.89

2.64

15.88

8.4

40.44

2.45

21.76

1.31

Haplic35.35
Chernozem

Humic Substances in Ecosystems
8, Šoporňa 13.-17.9.2009 Slovakia

Fig. 1. UV-VIS spectra

Q4/6 index (ratio A465/A665 nm)

Samples: humic substances extracts were prepared inmixture 0.1M sodium pyrophosphate and 0.1M NaOH
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Fluorescenceindex F
F=RFI (468/522) nm

Fig.2. SFS spectra at ∆λ=55 nm

Samples: humates were dissolved in MILI Q water,{522-55=467 nm}

c= 50 mg/l

Em.
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Exc.

Results
 All samples had main peak at emission

522 nm (∆λ=55 nm)
 Highest rel. intensity of fluorescence(RFI) at 522 nm had sample Haplic
Chernozem
 Lowest rel. intensity of fluorescence(RFI) at 522 nm had sample LepticCambisol
 Arable soil humate (E. Cambisol 1) had
higher RFI at higher wavelengths thangrassland soil humate (E. Cambisol 2)

Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Results and disscussion●Fig. 4 The dependence of fluorescence index on carbon contentof soil humates (4 samples), R=0.99
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Correlation

Fig.3 Correlation betweenfluorescence index and totalorganic carbon content ofsoil humates (4 samples),R=0.96●Fig. 5 The dependence colour index
on humus content of soil humates
Humic Substances in Ecosystems 8, (4Šoporňasamples),13.-17.9.2009
R=0.93
Slovakia

Conclusion


Optical properties are influenced by soil type andhumus fractional composition. In this study colour
indexes and fluorescence indexes by UV-Vis and SFS
methods were determined. The highest quality andhumification degree had Haplic Chernozem. The
lowest quality had Eutric Cambisol (arable).
From SFS measurement was evident that allsamples had the same main fluorophore atexcitation 467 nm a emission 522 nm (∆λ=55 nm).
The difference between the type of land use (arable,grassland) in the shape of the spectra was found.
Linear correlations between TOC and F, between Fand C content and between colour index Q4/6 andhumus content were determined.

Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Acknowledgement
This work has been supported by Grant Agency ofthe Czech Republic Project No.104/03/D135, by theProject NAZVA No. QH72039 and NAZVA QH81200.

Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

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Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

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Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Thank you for your attention!

Humic Substances in Ecosystems
8, Šoporňa 13.-17.9.2009 Slovakia