Fertilizers and soil conditioners -- Determination of humic and hydrophobic fulvic acids concentrations in fertilizer materials

This document specifies the procedure for the analysis of humic acids and hydrophobic fulvic acids which is applicable to dry and liquid materials used as ingredients in commercial fertilizers, soil amendments, and geological deposits.

Engrais et amendements minéraux basique - Détermination des acides humique et fulvique

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Published
Publication Date
09-Aug-2018
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6060 - International Standard published
Start Date
05-Jul-2018
Completion Date
10-Aug-2018
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INTERNATIONAL ISO
STANDARD 19822
First edition
2018-08
Fertilizers and soil conditioners —
Determination of humic and
hydrophobic fulvic acids
concentrations in fertilizer materials
Engrais et amendements minéraux basique - Détermination des
acides humique et fulvique
Reference number
ISO 19822:2018(E)
ISO 2018
---------------------- Page: 1 ----------------------
ISO 19822:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

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Published in Switzerland
ii © ISO 2018 – All rights reserved
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ISO 19822:2018(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative References ..................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principles ..................................................................................................................................................................................................................... 2

5 Warnings ....................................................................................................................................................................................................................... 2

6 Reagents ........................................................................................................................................................................................................................ 2

7 Apparatus ..................................................................................................................................................................................................................... 3

8 Preparing crucibles, drying, and weighing samples ....................................................................................................... 4

8.1 Preparing crucibles ............................................................................................................................................................................. 4

8.2 Drying and weighing solid analytical samples ............................................................................................................ 4

8.3 Drying and weighing liquid samples .................................................................................................................................... 5

9 Extraction procedure ....................................................................................................................................................................................... 5

10 Determination of ash content ................................................................................................................................................................. 6

11 Separation of Hydrophobic Fulvic Acids (HFA) ..................................................................................................................... 6

12 Hydrogen ion exchange ................................................................................................................................................................................. 7

13 Calculations................................................................................................................................................................................................................ 8

13.1 Determination of ash-free HA weight ................................................................................................................................. 8

13.2 Determination of ash-free HFA weight ............................................................................................................................... 8

13.3 Determination of % analyte in solid analytical samples ..................................................................................... 8

13.4 Determination of % analyte in liquid samples ............................................................................................................ 8

14 Resin regeneration and column preparation ......................................................................................................................... 8

Annex A (informative) Procedure to confirm the presence lignosulfonates ...........................................................10

Annex B (informative) ISO/CD 19822 interlaboratory study ..................................................................................................13

Bibliography .............................................................................................................................................................................................................................16

© ISO 2018 – All rights reserved iii
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ISO 19822:2018(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso

.org/iso/foreword .html.

This document was prepared by Technical Committee ISO/TC 134, Fertilizers and soil conditioners.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO 2018 – All rights reserved
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ISO 19822:2018(E)
Introduction

Humic substances are present in all ecosystems: oceans, rivers, lakes, and top soils. Quantifying the

amount of humic material present in these systems is essential for academic research and commercial

applications, specifically agricultural soil and plant management.

The increased use of humic substances in agriculture has generated intense interest among producers,

consumers, and regulators for a reliable method for quantification of the active ingredients in raw

humic ores and commercial fertilizer products; specifically humic and fulvic acids. As both commercial

trade and regulation of humic products are based on percentage (%) of the humic and fulvic acids in

commercial humic products, use of % units instead of SI units is warranted, therefore incorporated

into this standard.

This document establishes a method for the determination of humic acids (HA) and acidic hydrophobic

fulvic acids (HFA). The method is based on an existing preparative procedure use by the International

[1]

Humic Substances Society (IHSS) for extracting high purity HA and HFA from soil samples , which is a

[2]

modified form of the “classical” technique described in detail by Stevenson . The “classical” methods

and the IHSS method were developed as preparative methods for the fractionation of soil organic

matter; they were not intended to be used as quantitative analytical methods. The classical method of

extracting humic acids and fulvic acids from soil humus utilize a “strong base” to extract the alkaline

soluble materials, and then the alkaline extract solution is acidified to flocculate the humic acids, which

appear to precipitate out of solution. The remaining substances in solution after alkaline and acid

treatment were called fulvic acids.
This method modifies the “classical” technique in a number of ways:

— it determines the quantity of humic substances on an “ash free” basis (mineral salts excluded);

— the alkali extraction is done under anoxic conditions to reduce oxidation of the analytical sample

during extraction;

— it defines the materials that are soluble in both alkali and acid as the Fulvic Fraction;

— it can differentiate products containing certain non-humic materials that some manufactures claim

to contain humic substances;
[3] [4]

— it further defines HFA as materials of low sulfur content that bind to a hydrophobic resin at pH 1

[5][6]

, instead the classical, and perhaps more common definition, for fulvic acids as materials that are

defined as soluble in both acid and alkali solution. This stricter definition is necessary to distinguish

HFA from mineral salts, polysaccharides, amino sugars, amino acids, proteins, acids, and carbohydrates

[1][4]

that are extracted along with humic substances when using the “classical” method .

See Annex B for information on ISO/CD 19822 interlaboratory study.
© ISO 2018 – All rights reserved v
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INTERNATIONAL STANDARD ISO 19822:2018(E)
Fertilizers and soil conditioners — Determination of humic
and hydrophobic fulvic acids concentrations in fertilizer
materials
1 Scope

This document specifies the procedure for the analysis of humic acids and hydrophobic fulvic acids

which is applicable to dry and liquid materials used as ingredients in commercial fertilizers, soil

amendments, and geological deposits.
2 Normative References
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at https: //www .electropedia .org/
3.1
hydrophobic fulvic acids
HFA

materials composed of less than 0,75 % elemental sulfur (S) that are soluble in aqueous alkaline and

acid solution and are adsorbed at pH 1 onto a polymeric adsorbent resin of moderate polarity. The resin

is of a type designed for adsorption of amphiphilic compounds having molecular weights typical of

fulvic acids
3.2
fulvic fraction

alkali extracted portions of humic substances that are soluble in both alkali and acid aqueous solutions

3.3
humic acids

alkali extracted humic substances that are insoluble in strongly acidic solution and will precipitate

from the alkali extract in acid solutions of pH 1
3.4
humic substance

major organic constituent of natural organic matter consisting of complex heterogeneous mixtures

of carbon-based substances formed by biochemical reactions during the decay and transformation of

plant and microbial remains
3.5
lignosulfonates

amorphous light to dark brown powder or liquid derived from the sulfite pulping of softwoods. The

lignin framework is a sulfonated random polymer of three aromatic alcohols: coniferyl alcohol,

p-coumaryl alcohol, and sinapyl alcohol, of which coniferyl alcohol is the principle unit

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ISO 19822:2018(E)
4 Principles

4.1 This method determines ash-free quantities of HA and HFA gravimetrically after separation from

their matrix.

4.2 The method of extracting HA and HFA utilizes a strong base to extract the alkaline-soluble materials,

and then, after removal of non-soluble components, the alkaline solution is acidified to flocculate the HA.

4.3 The liquid supernatant remaining after the removal of the HA is called the Fulvic Fraction. The

Fulvic Fraction, which can contain Hydrophobic Fulvic Acids (HFA), is treated to determine the quantity

of HFA in the Fulvic Fraction by selective adsorption onto a methacrylic-ester resin designed to separate

the HFA from non-humic compounds. The material retained by the hydrophobic resin is referred to in the

[3]
literature as the hydrophobic acid fraction of soluble organic matter .
5 Warnings
5.1 Requirements
5.1.1 Good laboratory practices

Related standards (e.g. ISO/IEC 17025) should be followed at all times in regards to personal protective

equipment (safety glasses, handling strong acids, hydrochloric acid) and alkali (sodium hydroxide).

5.1.2 Moisture control

Humic and fulvic acids are hygroscopic materials; it is critical to prevent absorption of moisture during

the handling of dried materials.
5.3 Lignosulfonates

Lignosulfonates will damage the hydrophobic resin. This analytical method cannot differentiate

between hydrophobic fulvic acids and lignosulfonates, therefore pre-screening for the presence of

lignosulfonates is recommended for liquid products of unknown origin.
See Annex A.
5.4 Temperature control

Do not exceed 65 °C when drying the humic and fulvic analytes. The analytes are subject to

decomposition at higher temperatures.
6 Reagents

6.1 Sodium hydroxide solution, 0,1 M, dilute 3,99 g of 99,99 % purity NaOH in 1 l of deionized water.

6.2 Sodium hydroxide solution, 0,5 M, dilute 19,99 g of 99,99 % purity NaOH in 1 l of deionized water.

6.3 Hydrochloric acid solution, 6 M, dilute 12 M HCl with an equal part of deionized water.

6.4 Hydrochloric acid solution, 1 M, dilute 83,3 ml of 12 M HCl with 1 l of deionized water.

6.5 Hydrochloric acid solution, 0,1 M, perform a 1:10 dilution using 1 M HCl prepared in 6.4 with

final volume of 1 l using deionized water.
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ISO 19822:2018(E)
6.6 Nitrogen gas (UN1066) 99,9 % purity.

6.7 Methacrylic-ester resin, 40–60 mesh, approximately 0,79 ml·g pore volume, 225 Å mean pore

2 −1

size, 160 m ·g surface area, for adsorption of materials up to 150 000 MW, e.g. Supelite DAX-8 Resin, or

any other available resin meeting equivalent properties.
6.8 Amberlite IR-120 strong cation exchange resin, hydrogen form.
[8]
6.9 Deionized water .
6.10 Acetone.
7 Apparatus

7.1 Analytical balance with draft guard: capacity 210 g, with readability to ±0,000 1 g.

7.2 Drying oven, capable of 120 °C, precision ±3 °C.

7.3 Centrifuge, minimum relative centrifugal force 1 500 × g, capable of 3 900 × g.

7.4 4 ml to 50 ml or 250 ml polyethylene or HDPE centrifuge tubes, or heavy duty high

temperature resistant centrifuge tubes capable of 600 °C (for example; Kimble-Chase, catalog

number 45212-50 KIMAX).

7.5 4 ml to 100 ml wide-form crucibles (for example: Fisher Scientific catalog number FB-965-M).

7.6 Rotary evaporator 400 ml capacity.
7.7 Magnetic stir plates and 5 cm to 7 cm long magnetic stir bars.
7.8 pH meter and electrode.

7.9 Electrical conductivity meter with probe having a calibrated cell constant of approximately

one, as determined using standard protocols.
7.10 Spectrophotometer, capable of measuring ±0,005 absorbance units at 350 nm.
7.11 Peristaltic pump with a minimum flow rate of 1,2 ml·min and tubing.
7.12 Muffle furnace.
7.13 Rotating shaking mixer.
7.14 Desiccator with silica gel (or its equivalent) as desiccant.
7.15 Erlenmeyer flask, 1 000 ml.
7.16 Beaker, 4 l.
7.17 Graduated cylinder, 1 000 ml.
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ISO 19822:2018(E)
7.18 Glass chromatography column, 4 cm × 25 cm for DAX-8 resin.
7.19 Glass chromatography column, 5 cm × 60 cm for IR120 H+ exchange resin.
7.20 Ceramic mortar and pestle.
7.21 Sieve, 74 µm (#200 US mesh).
7.22 Parafilm® .
8 Preparing crucibles, drying, and weighing samples
8.1 Preparing crucibles

8.1.1 If using new crucibles, first wash them with acetone and then dry them in an oven at 105 °C for

2 hours.

8.1.2 Prepare previously used crucibles by washing in acetone, then firing them in a muffle furnace at

500 °C for 2 hours. Cool the crucibles in a desiccator to room temperature. Remove from desiccator when

cool, record weight of the crucibles to four decimal places.
8.2 Drying and weighing solid analytical samples

8.2.1 If the analytical sample is a solid material, crush and screen approximately 5 g of the analytical

sample to ≤75 µm making sure that the powder becomes well homogenized.

8.2.2 Transfer approximately 5 g analytical sample to a 100 ml crucible prepared according to 8.1.

8.2.3 Place the analytical sample in a drying oven for 24 h at a temperature of 62 ± 3 °C. (do not exceed

65 °C). If any clumping of the sample occurs during drying, break up the clumps with a glass rod. Continue

drying until the sample is dried to a constant weight. This can take up to 24 h;

8.2.4 After achieving constant weight, remove the analytical sample from the drying oven and

immediately place in a desiccator to cool.

NOTE Both humic and fulvic acids are hygroscopic materials, it is critical to prevent absorption of moisture

during the handling of these materials.

8.2.5 Weigh out approximately 2,5 g test portion from the dried analytical sample in a pre-weighed

crucible prepared according to 8.1, taking precautions to prevent moisture adsorption while handling.

Record the test portion + crucible weight to four decimal places. Proceed to 9.1 immediately or return

the crucible with test portion to the desiccator.

8.2.6 Determine the weight of the test portion by subtracting the weight of the crucible from the test

portion + crucible; record the result as the Test Portion Dried Weight.

1) Parafilm® is a trademark of Bemis NA, Neenah, Wisconsin, USA. This information is given for the convenience

of users of this document and does not constitute an endorsement by ISO of the product named. Equivalent products

may be used if they can be shown to lead to the same results. It is commonly used for sealing or protecting vessels.

It is a ductile, malleable, waterproof, odorless, translucent and cohesive thermoplastic.

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ISO 19822:2018(E)
8.3 Drying and weighing liquid samples

8.3.1 For liquid analytical samples, thoroughly homogenize the analytical sample by shaking the

sample for one minute in the container in which the liquid was delivered. Weigh out approximately

5 g test portion from the liquid analytical sample to four decimal places. Record this as the Liquid Test

Portion Weight.

For liquid analytical samples with an expected HFA content <1 %, use a test portion weight of 10 g.

8.3.2 Follow steps 8.2.2 to 8.2.6 for drying the sample and weighing the test portion of the sample.

9 Extraction procedure

9.1 From this point on, the method is the same for both solid and liquid samples.

Transfer the prepared test portion of the analytical sample to a 1 l Erlenmeyer flask containing a 5 cm

to 7 cm long magnetic stir bar. Add 0,1 M NaOH, constantly stirring on a stir plate, to a final volume of

1 l. Evacuate the head space with N and cover with parafilm (or seal the flask with a similar material).

Then mix vigorously on a stir plate (e.g. 300 rpm to 400 rpm). Stir liquid samples for 1 hour; stir the

solid samples for 16 hours to 18 hours.

NOTE For solid samples, it can be convenient to perform this step late in the day so the solid samples can be

stirred overnight.

9.1.1 After stirring, remove the flask from the stir plate, transfer to suitable centrifuge tubes and

centrifuge the entire volume at 3 900 × g for 30 minutes to separate any insoluble material from the

dissolved materials in the alkaline extract. Carefully transfer the alkaline extract to a clean 1 l Erlenmeyer

flask containing a magnetic stir bar. Discard the insoluble materials.

9.1.2 While gently stirring the solution, adjust the pH of the alkaline extract solution to flocculate

the HA from the acid soluble materials by adding 6 M HCl (1:1) drop-wise to the alkaline extract, until

1,0 ± 0,1 pH is reached.

9.1.3 Cover the flask with parafilm and mix for 1 h. After 1 hour, check the pH and readjust to

pH 1,0 ± 0,05 with additional 6 M HCl, if necessary. If the pH falls below 0,95, adjust the pH back to

1,0 ± 0,05 with 0,5 M NaOH solution. Continue mixing the acidified extract until it stabilizes at

pH 1,0 ± 0,05 for exactly 5 minutes. Do not let the acidified extract sit for longer than 5 minutes after the

pH stabilizes. Remove the pH electrode.
9.2 Separation of HA

9.2.1 Once the pH is stable, remove the flask from the mixer, and cover the flask with parafilm. Allow

the pH-adjusted extract to sit undisturbed for 4 hours ±5 minutes (do not exceed 4 hours). This stage is

critical to prevent further partitioning of the HA and HFA constituents. The flocculated HA will drop out

of solution.

9.2.2 Immediately centrifuge the extract solution at 3 900 × g for 30 minutes using pre-weighed 50 ml

centrifuge tubes to recover the flocculated humic acids portion. Decant the supernatant (Fulvic Fraction),

being careful not to include any of the flocculated HA. Typically, about 500 ml of the clarified extract can

be decanted without disturbing the flocculated HA. If hydrophobic fulvic acids (HFA) analysis is to be

performed, decant the fulvic fraction into a clean 1 l Erlenmeyer flask. [Alternatively: using heavy duty

high temperature centrifuge tubes instead of plastic centrifuge tubes eliminates the need to transfer the

flocculated HA to a crucible, decreasing labor and increasing precision.]
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ISO 19822:2018(E)

9.2.3 Centrifuge the tubes containing the flocculated HA again at 1,500 × g for 20 minutes to 30 minutes

to further separate the flocculated HA that appears to precipitate from the liquid fulvic fraction. If HFA

analysis is intended, add this supernatant to the decanted fulvic fraction supernatant in 9.2.2.

9.2.4 If using high temperature centrifuge tubes, place the tubes containing the flocculated HA in a

drying oven at 62 ± 3 °C. If using plastic or low temperature centrifuge tubes, carefully scrape all of the

flocculated HA from the centrifuge tubes into 100 ml wide-form crucible(s) prepared according to 8.1.

[8]

After scraping out the flocculated HA, add a minimal amount of deionized water to the centrifuge tube,

secure the caps on the tubes, shake the capped tubes vigorously to get all the flocculated HA out of the

tube, adding this deionized water/flocculated HA mixture to the crucible(s). Regardless of using either

heavy duty tubes or crucibles, dry the flocculate HA to constant weight (typically overnight) in an oven

at 62 ± 3 °C. Break up any clumps that form during drying using a glass rod, taking care to avoid removal

of any material from the tubes. Depending on the source of the HA, the drying process can take up to

24 hours.

NOTE The high temperature centrifuge tubes can be supported in the furnace using 50 ml beakers.

9.3.2 Once the flocculated HA is dried to constant weight, remove the tubes/crucibles from the drying

oven and immediately place them in a desiccator to cool. Once cooled to room temperature in the desiccator,

reweigh the tubes/crucibles containing the flocculated HA. Take precautions to reduce adsorption of

moisture during this step. Record the combined weight of the dried flocculated HA + tubes/crucibles,

subtract the weight of the tubes/crucibles; record this weight as the Dried Flocculated HA Weight.

10 Determination of ash content

10.1 At this stage, the dried flocculated HA contains some residual ash. The ash content is determined

by combusting the dried flocculated HA in either heavy duty centrifuge tubes or crucibles (prepared

according to 8.1) in a muffle furnace for 4 h at 500 °C. Wait until weight is constant. If any solid clumps

form during ashing, carefully break up the clumps using a glass rod. Do not use a metal rod.

10.2 After achieving constant weight, remove the tubes/crucibles containing the flocculated HA from

the muffle furnace and place them in a desiccator to cool to room temperature.

10.3 Once cool, determine the weight of the dried ash by subtracting the weight of the tube/crucible

from the combined weight of the tube/crucible + ash. Record the result as Flocculated HA Ash Weight.

11 Separation of Hydrophobic Fulvic Acids (HFA)

11.1 Hydrophobic fulvic acids (HFA) are separated from other acid soluble substances in the Fulvic

Fraction by selective adsorption to an acidified hydrophobic resin (i.e. Supelite DAX-8), to which

hydrophilic acid soluble components do not bind and can thus be removed. This is accomplished using

a 4 cm × 25 cm glass chromatography column partially filled with 280 ml of resin soaked in deionized

[8]

water to prevent air pockets. Maintain an optimum space of approximately 2,5 cm from the top of

the wet resin to the top of the column (see Figure 1). The intent is to leave sufficient space above the

resin to visually monitor the flow rate through the column. If using new resin, clean the resin prior to

use according to 14.1. After using the chromatography column resin, regeneration of the column resin

is performed according to 14.1.2. When finished using the resin at the end of the day, store in methanol.

[8]

Before reusing the resin, rinse the methanol from the resin with deionized water by using a slurry

technique in a large beaker until the resin is free of methanol (less than 2 mg·l dissolved organic

carbon). If the resin becomes discolored, or has been used about 15 times, perform a quality control test

6 © ISO 2018 – All rights reserved
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ISO 19822:2018(E)

using a quality control material. If necessary, clean the resin according to 14.1 or discard and replace

with new resin.

NOTE The fulvic fraction is in the Erlenmeyer flask on the right side of the photo.

Figure 1 — Example of apparatus used to separate hydrophobic fulvic acids from the fulvic

fraction

11.2 The Fulvic Fraction solution is passed through the column via the top of the column using a

peristaltic pump set at just enough pressure so that the flow rate is sufficient to completely cover the

resin in the column without overflowing the column or allowing the resin to be exposed to air. The

−1 −1.

suggested flow rate is about 4 ml·min to 5 ml·min . Do not allow the solution level to drop below the

top of the resin. Discard this effluent.
[8]

11.3 The column is then washed with deionized water using a peristaltic pump with enough pressure

−1 −1

so that the flow rate (about 4 ml·min to 5 ml·min ) is sufficient to maintain complete coverage of

resin. Continue washing until the UV absorbance A = 0,015 (350 nm) or after eluting 2 column volumes,

whichever occurs first. Do not exceed 2 column volumes. Discard the effluent.

11.4 The HFA is then desorbed from the resin by back elution (i.e. influent introduced into the bottom

of column) with 0,1 M NaOH, which is pumped using the peristaltic pump (flow rate about 4 ml·min

to 5 ml·min ). Capture the HFA-containing effluent. The HFA has been desorbed when the absorbance

of the effluent is A = 0,030 (350 nm) using a spectrophotometer or after eluting 3 column volumes,

whichever occurs first. Do not exceed 3 column volumes.
12 Hydrogen ion exchange

12.1 Using a hydrogen form H exchange resin prepared according to 14.2. Pour 500 ml of the prepared

H exchange resin into a 5 cm × 50 cm chromatography column according to 14.2.1. Pump the

...

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