ANALYSIS AND CHARACTERIZATION OF HUMICSUBSTANCES BY HPLC METHODS
Milan Hutta, Róbert Góra, Mária Chalányová, Radoslav Halko
Department of Analytical Chemistry, Faculty of Natural Sciences,
Comenius University

Mlynska dolina CH-2, 842 15 Bratislava, Slovakia
hutta@fns.uniba.sk
APVV-0595-07, VVCE-0070-07, VEGA-1/4474/07, VEGA-1/0870/09
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Introduction
Multicomponent analysis of
ionogenic
compounds
complex environmental(humic substances, etc.)
or biological matrices(biopolymers, low MWsubstances) is in many
aspects still problematic,
especially from thepoint-of-view of refinedrequirements of praxis.

Systematic combination of various separation principles and/or column separation
methods hyphenation offers many attractive features of manipulation withselectivity of separation and detection and speed of analysis
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Relation of sample complexity and theory of peakoverlap in separation systems.

No one current single analytical method can resolve
all components ofcomplex natural or technological samples as followsfrom
Davis and Giddings statistical peak overlap theory
based on homogeneous statistics

Davis J.M., Giddings J.C.: AnalChem., 55 (1983) 418.
J.M.Davis: Anal.Chem., 69 (1997) 3796.
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

What does it mean complex sample?
sample?
Each sample is complex at certain – usually low concentrationlevel of its components when number of sample components
exceeds component capacity of given separation or detection
method. Soil column chromatography.
(even highly purified water or rain water are complex samples at ppt and lower levels).

>10 ppm

1 ppm<0.1 ppm
Urine or manure RP-HPLC profile
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

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

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

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

RPRP-HPLC profiles of Peat Humic Acid on 30 nm pore diameter C18 column
Note: similar profiles are typical for almost every analytical method

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

RPRP-HPLC profiles of Peat Humic Acid on 30 nm pore diameter C18 column
Note: similar profiles are typical for almost all analytical methods

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

Basic aspects of combination of liquid chromatographymethods or alternative methods
For real sample pretreatment and multicomponent (trace)
analysis we should achieve whenever possible
 high selectivity of redistribution (HPLC-variable modes)
and detection
 high efficiency of transport phenomena (quantitative)and detection yield (sensitive, low LOD, LOQ) high enrichment (102-106) and pre-concentration factors
achievable in gradient and column switching HPLC quantitative transfer of sample within analyticalprocedure and/or method
 accurate results short analysis duration (fast and ultrafast LC 1-10 min) low cost of analysis
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

This is practically impossible without complex and complicatedanalytical procedure schema, smart and expensive instrumentationand consistent analyses quality control.
Definite effort is given to elaboration of robust, validated anduniversally applicable analytical methods based also on HPLC.
HPLC.

In spite of fact thatsystem of quality assurance and quality control
suffers fromlack of proper certified HSs standard reference materials
refering to certain exact locality
for validation of both existing and new analytical methods
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

COMBINATION OF SEPARATION TECHNIQUES FORANALYSIS OF IONOGENIC SUBSTANCES INCOMPLEX MATRICES
Complexity of problems associated with trace or ultratrace analysis (ppbto ppt) of substances in a natural matrices is reflected mainly by the fact
thatfor their successful solution we need to combine or hyphenate even highly
efficient techniques. Beside on-line coupled separation and identification
or detection methods, e.g., HPLC-MS etc., possibilities of combinations
and hyphenation of separation methods is intensively investigated andresearched.
This originates so called two- or multi-dimensional separation
techniques, e.g. HPLC-HPLC or ITP-CZE and the other.

These methods are according to their principles applicable for
distinct groups of substances having differing numbers ofentities.
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

By the combination of analytical methods we canaccording to the nature of problem and aim of theproblem solution achieve that number of analysed
analysed
substances:
 increases for methods complementary each other (someLC-LC methods, e.g. SEC- RPLC) stays unchanged, but the combination enables bettercharacterization of sample and/or identification ability ofsample components (e.g. PyGC-MS, IEX-RPLC-MS etc.)
by increase of information content of analysis, decreases when such methods are combined, that first
method has narrower applicability than the second one, but
their combination gives higher discrimination ability.

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

CONCEPTS OF METHODS AND TECHNIQUESCOMBINATION
Systematic change of selectivity during separation process enables us
to attain practically not restricted separation possibilities under
the condition that we are not restricted by available time.
Advantages of successive (tandem) use of various separation modes (modesequencing) we can express mathematically. According to Giddings is peakcapacity of simple isocratic mode φ given by equation:

φ = 1 + (+1/2 / m ). ln( 1 + k´n )where for unity resolution

m = 4 (4 σ separation),
N = plate number,
k'n = retention factor of last peakin a series of peaks numbered from zero (nonretained) to n (last detectablepeak).\. Peak capacity φ is based on determination of maximal number of solutes (peaks), that can be resolved with unity
resolution RS = 1 in a given system and within certain time interval

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

Peak capacity φHPLC gradient elution
HPLC isocratic elution
SEC isocratic elution

200 - 300
cca 50
3-10CZE
ITPIEF

200 – 800
cca 100
200 – 300

If peak capacity of all n modes is equal, we get:

φT = φ n
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Requirement of nonnon--redundancy,redundancy, or nonnon--correlationand ororththogonality
ogonality between individual combinedmodes is the main requirement and at the sametime also assumption or premise of the relation

φT = φ nbecause on contraryCombination of n equal modes(identical columns) in aseries increases overall efficiency according to equation:
φT = 1 + [(n+) 1/2 / m ]. ln(1 + k n )what is approximately equal toφΤ ~ n 1/2 .φφiHumic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Requirement of nonnon--redundancy,redundancy, or nonnon--correlationand ororththogonality
ogonality
Orthogonality of separations or detection is a discussed
philosophy of multi-dimensional system design.
Orthogonal design means that features multiply each other'sutility, and overlap as little as possible.
Given set of carefully-crafted orthogonal features can enable
a wide variety of possibilities for complex analysis throughcombining those features.
In theory, this means that one can have a small and simple
instrumental base, which enables an extremely richmethodological environment for chemical analysis.
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

The highest separation efficiency is achieved bydiscrete combination of selective methods.
For proper selection of the most effective combinations we shouldtake into account that correlation of the data is not given only byphysico-chemical principle of combined methods, but also by asample itself
chemical structure of components
number of sample components

and favorites proper choice ofdifferently selective – focused to dissimilar sample properties,
high performance andvery compatible separation processes.
Multicolumn switching Liquid Chromatography
Liquid Chromatography and Capillary Electrophoresis
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Criteria for selection of methods suitable for combining in analyticalseparation schema applied to methods of liquid chromatography
and capillary electrophoresis

Efficiency,, Selectivity, Compatibility
Efficiency
considerations
HPLC
Efficiency 1000 to 10000 plates per column. Exceptionally 1 - 2
orders of magnitude higher
Peak capacity – for common standard columns (250mm/4mm,3-5µµm)
isocratic 50-100, gradient 250-300, SEC 5 to 20
CZE
Efficiency 5000 to 500 000, peak capacity 300 - 500 for low molecularweight substances and 500 to 800 for polymeric substances.
ITP
peak capacity equivalent -"zone capacity" is estimated to50 - 100 .

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

Search for orthogonal combinations of RPHPLC and cITP
Selectivity
Correlation of retention factors k (RP HPLC) and relative
step heights RSH Abbr.
(cITP) of 8 randomly selected k´N-basic
compounds

Compound1

o-toluidine

2,10y = -0,014x + 0,5307
2
R1,20= 0,0122

OTO

0,9

aniline0,7p-toluidine

triethylamin
pyridine

0,6
RSH

2,4,6-trimethylpyridine

A+I

0,8RSH0,9381
0,7797PTO

2,900,6384

TMP

7,100,4521

TEA

1,100,4237

PYR

1,900,3446

APY5,300,2825

0,50,40,30,22-aminopyridine
diethylenetriamine

0,100,00

Separation conditions:
HPLC - Separon SIX C18, CGC 5um, MeOH / H2O

DEA
2,00

4,00
kcITP-

1,10
6,00

0,06788,00

LE: ammonia 10mM , acetate 15mM, HEC, pH=5,2
TE: acetic acid 10 mM, pH=5Hutta M.,
Kaniansky
D., Zelenska
V. unpublished results
Humic Substances in Ecosystems
8, Šoporňa13.-17.9.2009
Slovakia

Search for orthogonal combinations of LC and CE
Selectivity at High CompatibilityCorrelation of qualitative parameters ofRPHPLC vs CZE, 18 N bases

retention time RPHPLC(min)imidazole
pyridinecreatinine
quinineanilinetrimethylpyridine
cytozineo-toluidine
nicotinamide
adeninehistidineN-1naphthtylethylenediamine
4-aminophenazone
cytidineadenosine
tryptophanetyrosine
p-aminoacetophenone

7

y = 0,2361x + 1,2964

6

2

R = 0,1505

543
2
1005
10
migration time CZE (min)Separation conditions:
HPLC - Chromolith Performance RP18e, MeOH / pH 2.5 buffer

CZE - background electrolyte , pH 2.5Humic Substances in Ecosystems 8,HuttaŠoporňaM., Mamrošová
M.,Ondrejášová B . unpublished results
13.-17.9.2009
Slovakia

15

Search for orthogonal combinations of LC and CE
Selectivity
Correlation of retention factors k (RPHPLC)
and relative step heights RSH (cITP)
Abbr.
acids
of C1 to C10 n-fatty

Compoundk´RSH

1C10,290,156

Acetic acid

0,9C20,290,311

Propionic acid

0,8C30,350,405

Butyric acid

0,7C40,410,5220,820,606

1,650,685

C7y = 0,1417Ln(x) + 0,5335 3,110,728Valeric acid
Hexanoic acid
Heptanoic acid
Octanoic acid
Nonanoic acid
Decanoic acid

RSHFormic acid

0,6C5 y = 0,0372x + 0,4527
20,5C60,40,3R = 0,5448

2R = 0,835

5,440,767

C99,500,815

C1013,400,844

C80,20,100,00

5,00

HPLC Purospher RP C18e, Methanol-water, pH 2.0

10,00

ITP

15,00
kLE, TE

Hutta M., Kaniansky D., Kovaľ M., unpublished results
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Discrete vs continual
In principle we can combine two methods as discrete methods, 2D separation then exploit greater
separation space in which every and each sample
component has its own exact coordinates continualmethods, 2D separation requires instantpresence of both separation mechanisms and movementof separated components we can imagine as trajectoriesof points in 2D space, what restricts available separation
space (2 D Planar LC, CEC, MEKC)

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

Compatibility and selectivity considerations– microbore
microbore,, liquid (mobile, carrier
carrier,, electrolyte) phase
c ITP

CZE

Diluting or
Diluting orconcentrating
concentrating

Concentrating

Diluting

Reversed Phase
Size exclusion
Ion Exchange
HILIC
Hydrophobic
Interaction
Affinity IMAC

pKpK

Effective mobility infully charged state

Effective mobility
in fully chargedstate

HPLC

Normal Phase

HPLC

Reversed Phase
Size exclusion
Ion Exchange
HILIC
Hydrophobic
Interaction
Affinity IMAC
Normal Phase

Secondary equilibria
Complexation

Secondaryequilibria

Inclusion

Complexation

Solvation

Inclusion

Counterion

Solvation
Counterion

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

ANALYSIS AND CHARACTERIZATION OF HUMIC SUBSTANCESISOLATED FROM PEAT AND SOIL USING OFFOFF-LINE COMBINATION
OF LIQUID CHROMATOGRAPHIC METHODSRóbert GóraGóra,, Pavol RohárikRohárik,, Milan Hutta

SEC,Separon
HEMA100,
DMF/buffer
pH 3.00

RP-HPLC C18, DMF/buffer pH 3.00
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

IMAC
UTILIZATION OF IMMOBILIZED ALUMINIUM(III)ALUMINIUM(III) METAL ION AFFINITY
CHROMATOGRAPHY FOR ANALYSIS OF HUMIC ACID
Radoslav HalkoHalko,, Tibor NeuročnýNeuročný,, Milan HuttaStructure of chelate sorbent Iontosorb SALICYL – Al(III)

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

Coupling and Interfacing
Four levels of coupling can be distinguished : off-line (manual),
 at-line (robotic interface) on-line (coupling via a transfer line) in-line (complete integration including sample preparation)

Heart-cutting (non-comprehensive) or comprehensive
sampling from first to second step or dimension
Valcárcel M. et al. 2001
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

ONON-LINE FLOW
FLOW--THROUGH EXTRACTION–
EXTRACTION–PRECONCENTRATIONPRECONCENTRATION--LARGE VOLUMEINJECTION REVERSEDREVERSED-PHASE LIQUID CHROMATOGRAPHY METHOD FOR TRACE
DETERMINATION OF SELECTED PYRETHROIDS IN SLOVAK SOIL MICROMICRO-SAMPLESSAMPLES
Mária Chalányová
Chalányová,, Milan Hutta
Hutta,, Martin Pagáč

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

3D
SEC (6 hours) – RPLC (6 minutes) – CE (2 seconds)

Moore, Jorgenson
1995
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Interfacing HPLC and ITP - CZE
RP HPLCc ITPXHutta M., Gora R. 2003=?

Nagyova I., Kaniansky D., 2001

Orthogonal systems for humic substances characterisation ?
Humic Substances in Ecosystems 8, Šoporňa 13.-17.9.2009 Slovakia

Future trends ?

 Search for orthogonal systems
 3 D HPLC-HPLC-ITP-CZE – (DAD, 3D

FLD, ELSD, EC Arrays, MSn…)
 Fast selectivity tuning 1-3 min per method

Past D

Present DD

Future DDD

and more DDDD…

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