lỉrciíctiKnMs

DETERMINATION OF HUMIC
AND FULVIC ACIDS IN FERTILIZERS BY
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY - SIZE EXCLUSION
CHROMATOGRAPHY

• NGUYEN HOANG DOM - DANG BAO TRUNG

ABSTRACT:
This study aims to develop a quantitative method of humic and fulvic acids in commercial
fertilizer samples by using the high-performance liquid chromatography size exclusion
chromatography (HPLC-SEC) with the use of UV detector. In comparison with the Walkley-Black
method which uses a redox titration (TCVN 8561:2010), this method eliminates the chemical
interferences caused by the oxidation of the organic impurities (positive eưors in determination of
carbon contents). Furthermore, the size exclusion chromatography technique is able to measure the
molecular weights and their distribution of such the humic substances. In accordance with the
Association of Official Analytical Chemists (AOAC) and the EURACHEM, the method validation is
assessed via selectivity, method linearity, limit of detection (LOD) and limit of quantification (LOỌ),
precision (repeatability and reproducibility) and trueness (recovery), and measurement uncertainty.
Keywords: humic acids, fulvic acids, fertilizer, size exclusion chromatography.

1. Introduction HA and FA are chelators, combining with

Humic substances are the major organic
components of soil, involving humic acids, fulvic
acids and humins [1]. Humic acids (HA) have
complex structures, containing ionic groups to
form chelate complexes, characterized by the
ability to restrain and release metal cations in a
plant’s grow environment. Humic acids are
aqueous-soluble at pH > 2. Meanwhile, fulvic
acids (FA) are types of HA with lower molecular
weights but higher oxygen contents, which can
absolutely dissolve in water regardless of the pH
values. Concerning soil fertility and crop yield,
minerals to produce organic compounds that can
be efficiently absorbed via the crop’s roots. In
addition, they also help the soil retain water and
restrict the intrusion of toxins toward crops.
Although HA and FA are not fertilizers, they are
excellent fertilizer carriers and activators. It was
evidenced that foliar fertilizers containing such
types of acids exhibited to be up to 5 times more
efficient in comparison with similar fertilizers. It
is important to time-fertilize with the appropriate
HA and FA contents, in order to activate growth,
flowering and fruit set.

430 SỐ 14 - Tháng 6/2021


In agricultural production, the increasing use
of humic substances is demanding the
quantification methods of HA and FA in raw
materials and fertilizers. However, HA and FA do
not have well-defined and consistent chemical
structures; therefore, an accurate quantitative
method seems to not exist. Currently, commercial
laboratories have offered three methods to
determine the content of HA or their derivatives,
based on the solubility of HA in dilute alkaline
solutions and their precipitation after acidification
[2]: (i) colorimetric method developed by
Mehlich (1984) [3]; however, FA and other
components (amino acids, proteins, sugars, fatty
acids, etc.) dissolved in alkaline cannot be ruled
out at the characterized wavelength; therefore,
HA should be precipitated at pH < 2 and re­
dissolved before colorimetric analysis; (ii) a
semi-quantitative method developed by the
California Department of Food and Agriculture
(CDFA) [4], based on the precipitation of HA at
pH < 2 and analyzed by gravimetric method;
therefore, the positive error caused by the ash
content cannot be eliminated; (iii) an improved
method described by Swift (1996) [5], which is
f>ased on the precipitation of HA at pH < 2 and
heir purification by a couple times washing in
TC1/HF solution. In Vietnam, HA and FA are
determined based on Vietnamese National
Standards, involving TCVN 11456:2016 (in soil)
[6] and TCVN 8561:2010 (in fertilizers) [7], by
Walkley-Black method applying a redox titration
(axidation of organic compounds by K2Cr2O7 in
I12SO4 medium and then back-titration of the
excess K2Cr2O7 by Fe2+ solution). The principle is
c anstructed on the solubility of both HA and FA in
a Ikaline media, determining the total HA and FA;
and the insolubility of HA in acidic media and
tl us deduce the only FA content.
This study aims to develop a quantitative
method of HA and FA in commercial fertilizer
samples, using high performance liquid
chromatography - size exclusion chromatography
(I- PLC-SEC). HPLC-SEC is a frequently used
technique to separate dissolved organic matters,
without pre-extraction of samples; therefore,
adrpting quick response, high sensitivity and
reproducibility, and widespread availability [8], In
HÓA HỌC-CÔNG NGHỆ THựC PHẨM
particular, HPLC-SEC with a uv detector permits
to estimate analyte’s size and the corresponding
distribution, including HA and FA in fertilizer
samples. In reality, the HA and FA ingredients of
raw materials are unknown, and their structures are
not well-defined and consistent; therefore, the
quantification of HA and FA developed by HPLC-
SEC is verified via the Walkley-Black method, in
accordance with the Association of Official
Analytical Chemists (AOAC) and the
EURACHEM.
2. Experimental
2.1. Exfraction of HA and FA in raw materials
andfertilizer samples
After homogenization and pulverizing (using
0.2 mm mesh sieve), a mixture of about 5 g raw
material (or fertilizer samples) and 100 mL
solution of sodium hydroxide/sodium
pyrophosphate (pH 13) was stirred for 1 hour and
stood overnight. After filtration, the resulting
solution was adjusted to pH 7 with 1 N H2SO4
solution and marked to 200 mL solution with
distilled water, to produce the solution A.
In 50 mL solution A, HA was precipitated by
adjusting pH to 1 (with 1 N H2SO4) and heating for
1-2 hours. After filtration, the resulting solution was
marked to 100 mL and then diluted 5 times by
distilled water (namely solution F, containing FA).
The precipitate was dissolved in the phosphate
buffer solution (pH 6.8) and 0.025 M Na2SO4, the
resulting solution was marked to 100 mL and then
diluted 25 times by distilled water (namely solution
H, containing HA).
2.2. Quantification of HA and FA by Walkley-
Black method
A mixture of 5 mL solution H (or solution F), 5
mL of 0.2 N K2Cr2O7 solution and 20 mL of
concentrated H2SO4 solution was shortly shaken
and then stood for 30 min. Afterward, 50 mL
distilled water and 5 mL of concentrated H3PO4
solution were added into the solution and cooled at
room temperature. A drop of ferroin indicator was
added and then the excess K2Cr2O7 was back
titrated with Mohr solution of 0.1 N Fe2+ until the
red end point. The blank samples were performed
with the same procedure. The concentrations of HA
or FA converted as carbon contents (mg L1) were
calculated as the equation 1.
So 14-Tháng 6/2021 431
TẠP CHÍ CÔNG THƯƠNG
C(mgL-‘) = (A-B)
^K2Cr2O7 x N|<2Cr2O7 (1)
x A
3 DF
X—A-X-HT- X 1000
0.75 VH/F
Where: A and B = volumes (mL) of the standard
Mohr solution titrated with the blank sample and
the measured solution; VK2Cr2Q7 and NK2Cr2Q7 =
volume (mL) and normality (N) of K2Cr2O7
solution; 3 = equivalent weight of carbon; 0.75 =
75% carbon oxidized by K2Cr2O7; DF = dilution
factor; VH/F = volume (mL) of solution H (or
solution F).
2.3. Determination of HA and FA by HPLC-
SEC
After filtration with PTFE membrane filter
0.45 pm pore size, 20 pL solution H (or solution F)
was injected into HPLC system (Agilent 1290
Infinity II LC), using UV/DAD (1290 Infinity II
Diode Array Detector) detected at wavelength of
240 nm, a BioSep-SEC-s2000 column
(Phenomenex BioSep™, 300 X 7.8 mm, 5 pm),
with a phosphate buffer solution (pH 6.8, 0.025 M
Na2SO4) in Milli-Q water as a mobile phase. The
flow rate was 0.5 mL min1 and the operating time
was 30 min.
Construction of calibration curves of HA and
FA: the solution H (or the solution F) was diluted
by phosphate buffer solution, with variable carbon
concentrations of 0-180 mg L'1 (for HA) and 0-20
mg L 1 (for FA). The concentrations of HA or FA
converted as carbon contents (%c, w/w) were
calculated as the equation 2.
%c = -Ịễ?^-x VH/p X ———— X 100(2)
lOOOxa H/F m v
Where: s = HA or FA chromatographic peak
areas; a and b = slope and y-intercept of the
straight-line calibration curves; VH/F = volume (L)
of sample H (or sample F); DF = dilution factor;
m = mass of initial sample.
The number average molecular weight (Mn),
the weight average molecular weight (Mw) and the
polydispersity index (PDI) of HA and FA were
estimated on Agilent GPC Rev B.01.02 data
analysis software, based on standard curve of
polyethylene glycol’s molecular weight versus
retention time.
432 So 14-Tháng 6/2021
3. Results and Discussion
3.1. Sample preparation and determination of
HA and FA by HPLC-SEC
In fertilizer production, commercial ingredients
of HA and FA are generally unknown, and their
structures are ill-defined and inconsistent.
Therefore, the standard HA and FA solutions were
performed by extraction of such the humic
substances in raw materials and then quantitatively
analyzed by Walkley-Black method. In fact, the
HA and FA samples were absolutely dissolved in
highly alkaline solution; in particular
pyrophosphate can form the complexes with
calcium ions and other transition metal ions like
iron or copper, eliminating free HA and FA forms
and thus their highly solubility in alkaline media
[9], On the other hand, HA could be separated by
their precipitation in an acidic medium (pH 1) and
then their re-dissolving in a high pH solution. The
HA and FA concentrations were separately
determined by a redox titration method, and then
calculated following the equation 1. The
corresponding standard solutions were prepared
with variable carbon concentrations of 0-180 mg L1
(for HA) and 0-20 mg L1 (for FA) for the further
calibration.
In this study, HA and FA were analysed by
HPLC-SEC, using a DAD detector with the
wavelength range of 200-400 nm. The absorption
spectra of HA, FA and the blank sample showed
the maximum absorbances at the range of 230-240
nm and no interfering absorption caused by the
mobile phase with the wavelengths greater than
240 nm (Figure 1), resulting in the wavelength
chosen at 240 nm. The mobile phase containing a
phosphate buffer solution (pH 6.8, 0.025 M
Na2SO4) in Milli-Q water was optimized with the
flow rate of 0.5 mL min1, giving the good
performance parameters involving the maximum
intensities, the symmetric peaks in the range of
0.5-1.5 (tailing factor: Tf . 0.9 for HA and Tf= 1.5
for FA), short run time (retention time: RT = 20.9
min for HA and RT = 21.1 min for FA) (Figure 2).
Note that HA and FA were separately analysed
from the solutions H and F due to the restriction of
column availability, the resolution factor can be
ruled out.
 HÓA HỌC' CÔNG NGHỆ THựC PHẨM

In comparison with the redox titration method, Figure 1. Spectra of HA, FA and the blank
HA and FA can be quantitative analysed by HPLC- sample In a phosphate buffer solution (pH 6.8,
SEC, eliminating the chemical interferences 0.025 M Na2SOj) in water as a mobile phase,
caused by the oxidation of the organic impurities at the wavelength scanning of200-400 nm.
(positive errors in determination of carbon
contents). Furthermore, size exclusion
chromatography technique permitted to measure
the molecular weights and their distribution of HA
and FA based on standard curve of polyethylene
glycol’s molecular weight versus retention time
(Figure 3 and Table 1), which affect their mobility
and nutrient regulation in soil [10], Polyethylene
glycol is non-ionic polymer, meanwhile HA and FA
are multi-charged species in neuttal media.
Therefore, their hydrated ions appeared result in a
change of their molecular weight - size relationship
in comparison with polyethylene glycol. In order to Soure: Authors
minimize this effect, the viscosity decreasing and
the ionic strength increasing could be occurred by Figure 2. Chromatographs of the standard HA
adding 0.025 M Na2SO4 in the mobile phase. The (solution H) and FA (solution F) at 240 nm.
polydispersity index of HA (PDI = 2.189) is larger
than that of FA (PDI = 1.454), indicating the
broader molecular weight of HA. In fact, the size
and the molecular weight of HA and FA are
lynamic with regard to concentration, pH, ionic
strength and counter-ions; however, such the
characteristics were obtained with the different
Ỉ suits applying the different methods [10]. In this
-idy, the commercial standard HA and FA
urchased from Aldrich Sigma) showed the
c ifferent molecular weights and their distribution in Soure: Authors

igure 3. Molecular weight distribution of HA (solution H) and FA (solution F)

Molar mass [D]

Soure: Authors

SỐ 14-Tháng 6/2021 433

TẠP CHÌ CÔNG THƯƠNG

comparison with HA and FA isolated from raw
materials (solutions H and F). Therefore,
quantitative analysis of HA and FA by HPLC-SEC
should be carried out with the calibration of HA and
FA isolated from corresponding raw materials.
(i) Selectivity: the standard HA and FA
solutions and the blank sample were injected into
chromatography system for 6 replicates. In
contrast to the signal absence during 30 min for all
the blank samples, the HA peak (RT = 20.9 min)

Table 1. The number average molecular weight (Mn), the weight average molecular weight (Mw)
and the polydispersity index (PDI) of HA and FA

Entry Parameters HA FA

1 The number average molecular weight (Mn, Da) 1710 1081

2 The weight average molecular weight (Mw, Da) 3743 1572

3 The polydispersity index (PDI = MỰIVỤ 2.189 1.454

Soure: Authors

3.2. Method validation
The method validation was performed (fit for
intended use) on the solutions standardized from
raw materials (172.0 mg/L solution HA, 98.0 mg/L
solution FA and the blank sample), involving (i)
selectivity, (ii) method linearity, (iii) limit of
detection (LOD) and limit of quantification (LOQ),
(iv) precision (repeatability and reproducibility)
and trueness (recovery), and (v) measurement
uncertainty, in accordance with Association of
Official Analytical Chemists (AOAC) and
EURACHEM, summarized in Table 2.
and the FA peak (RT = 21.1 min) were always
observed from the corresponding standard
solutions.
(ii) Method linearity: the standard HA and FA
solutions (11 solutions for each substance) with
variable carbon concentrations (mg L'1) were
injected into chromatography system and the
calibration curves were constructed by plotting
the peak areas of various carbon concentrations.
The method was found to be linear in the
measured ranges with the strong correlation
coefficients (r2 > 0.99).

Table 2. Validation parameters for HA and FA analysis

Entry Parameters HA FA

1 Linearity range (mg L’1) 8.6-172.0 0.98-19.60

2 Linear equation y = 69.49x- 625.5 y = 36.95x-9.319

3 Correlation coefficient (r2) 0.9990 0.9977

4 Limit of detection, LCD (mg L’1) 1.27 0.49

5 Limit of quantification, LOQ (mg L’1) 4.23 1.63

6 Repeatability, RSDr (%) 4.8 (< 5.3) 5.6 (<7.3)

7 Reproducibility, RSDr (%) 2.4 (< 8) 3.8 (< 11)

8 Recovery (%) 93.3(90-107) 90.7(80-110)

9 Combined standard uncertainty, uc (%) 7.1 10.1

10 Expanded uncertainty, ue (%) 14.2 (< 16) 20.2 (< 22)

Soure: Authors

434 So 14 - Tháng 6/2021

 HÓA HỌC-CÔNG NGHỆ THựC PHẨM

(iii) Limit of detection (LOD) and limit of
quantification (LOQ): the blank samples spiked
with known concentrations of humic substances
were analysed to reach the signal to noise ratios of
S/N - 3 (LOD, 6 replicates) and S/N =10 (LOQ,
12 replicates).
(iv) Precision (repeatability and reproducibility)
and trueness (recovery): the standard HA and FA
solutions were analysed to estimate the relative
standard deviation of repeatability (RSDr,
performed with 6 replicates by the same personnel
at the same times) and the relative standard
deviation of reproducibility (RSDr, performed with
10 replicates by different personnel at different
times). Recovery test was performed on the spiked
samples, showing the values in the acceptable
recovery ranges.
(v) Measurement uncertainty: characterizing
the dispersion of the values that could reasonably
be attributed to the measured value. Combined
standard uncertainty (Uc) was estimated via
reproducibility and recovery; and expanded
uncertainty (Ue) was estimated at the 95%
confidence level and the coverage factor of 2,
indicating the acceptable values.
4. Conclusions
A reliable method was developed with the aim to
analyze humic and fulvic acids in commercial
fertilizer samples by high performance liquid
chromatography - size exclusion chromatography
(HPLC-SEC), using a uv detector with the
wavelength of 240 nm. Using a phosphate buffer
solution (pH 6.8, 0.025 M Na2SO4) in water as a
mobile phase with the flow rate of 0.5 mL min1
offered the good performance parameters involving
the maximum intensities and the symmetric peaks.
In particular, the molecular weight distribution of
such the acids was also estimated, explaining the
irrelevant and inconsistent results from various
methods. Therefore, an analytical method should be
developed and applied to the particular samples of
such the humic substances. The method validation
was well-performed in accordance with AOAC and
EURACHEM ■

Acknowledgments:
We acknowledge the support of time and facilities from Ho Chi Minh City University of Technology
(HCMUT), VNU-HCM and Petro Vietnam Camau Fertilizer Joint Stock Companyfor this study.

REFERENCES:

1. R. Pettit. (2008). Organic Matter, Humus, Humate, Humic Acid, Fulvic Acid and Humin: Their Importance in Soil
Fertility and Plant Health. Texas A&M University, 1-24.
2. R. T. Lamar, K. H. Talbot. (2009). Critical Comparison of Humic Acid Test Methods. Communications in Soil
Science and Plant Analysis, 40,2309-2322.
3. A. Mehlich. (1984). Photometric determination of humic matter in soils: A proposed method. Communications in
Soil Science and Plant Analysis, 15,1417-1422.
4. California Department of Food and Agriculture (CDFA). (1999). Humic acid method. Available from CDFA
Fertilizing Materials Inspection Program: 916-445-0444.
5. R. s. Swift. (1996). Organic matter characterization in Methods ofsoil analysis, part 3: Chemical methods (ed. D. L.
Sparks), 1011-1069. Madison, Wise.: Soil Science Society of America.
6. Ministry of Science and Technology of Vietnam. (2016). Vietnamese National Standards (TCVN 11456:2016).
Soil quality - Determination of humic acid and fulvic acid by Walkley-Black method.
7. Ministry of Science and Technology of Vietnam. (2010). Vietnamese National Standards (TCVN 8561:2010),
Fertilizers - Method for determination of humic acid and fulvic acid.
8. J. J. Alberts, M. Takács, p. K. Egeberg. (2002). Total luminescence specttal characteristics of natural organic
matter (NOM) size fractions as defined by ultrafiltration and high performance size exclusion chromatography
(HPSEC). Organic Geochemistry, 33,817-828.

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9. H. Kipton, J. Powell, R. M. Town. (1992). Solubility and fractionation of humic acid, effect of pH and ionic
medium. Analytica Chimica Acta, 267,47-54.
10. M. Klucáková. (2018). Size and Charge Evaluation of Standard Humic and Fulvic Acids as Crucial Factors to
Determine Theứ Envứonmental Behavior and Impact. Frontiers in Chemistry, 6,235.

Received date: May 3,2021

Reviewed date: May 17,2021
Accepted date: June 12,2021

Author’s information:
1. NGUYEN HOANG DOM w
2. PhD. DANG BAO TRUNG **
'Faculty of Chemical Engineering, Ho Chi Minh City University of Technology,
Vietnam National University Ho Chi Minh City
2PetroVietnam Camau Fertilizer Joint Stock Company

XÂ Y DựNG QUY TRÌNH ĐỊNH LƯỢNG AXIT HUMIC

VÀ AXIT FULVIC TRONG PHÂN BÓN BANG PHƯƠNG PHÁP
SẮC KÝ RÂY PHÂN TỬ HỆU NĂNG CAO

• NGUYỄN HOÀNG ĐỞM'2

• TS. ĐẶNG BẢO TRUNG1*
1 Khoa Kỹ thuật Hóa học, Trường Đại học Bách Khoa,
Đại học Quốc Gia TP. Hồ Chí Minh
2 Công ty cổ phần Phân bón Dầu khí Cà Mau

TÓM TẮT:
Mục đích của nghiên cứu nhằm phát triển một phương pháp định lượng axit humic và axit fulvic
hong cấc mẫu phân bón thương mại bằng kỹ thuật sắc ký rây phân tử hiệu năng cao (HPLC-SEC)
với đầu dò uv. So với phương pháp Walkley-Black - ứng dụng kỹ thuật chuẩn độ oxy hóa - khử
(TCVN 8561:2010), phương pháp này loại trừ các yếu tố nhiễu hóa học gây ra bởi sự oxy hóa các tạp
chất hữu cơ khác (dẫn đến sai số dương ưong việc xác định hàm lượng cacbon tổng). Hơn nữa, kỹ
thuật sắc ký rây phân tử cho phép xác định sự phân bố khối lượng phân tử của các hợp chất humic
này. Theo tiêu chuẩn của Hiệp hội các nhà hóa học phân tích chính thức (AOAC) và EURACHEM,
việc xác nhận giá trị sử dụng của phương pháp được tiến hành bao gồm độ chọn lọc, khoảng tuyến
tính, giới hạn phát hiện (LOD) và giới hạn định lượng (LOQ), độ chụm (độ lặp lại và độ tái lập) và độ
đúng (độ thu hồi), và độ không đảm bảo đo.
Từ khóa: axit humic, axit fulvic, phân bón, sắc ký rây phân tử.

43Ó SỐ 14-Tháng 6/2021