CERTIFICATE

Mobile Phase: 5mM Sulfuric acid

Flow Rate [rnl/min): 0,65
Temperature: 35
Pressure [bar): 30
Detection: UV, 210 nm
Injection Volume [1-11): 10.0

Ret.Time
[min) I 80
1 Oxalic acid 5,49
2 Citric acid 6,97
3 Malic acid 8,35
4 Succinic acid 10,50
5 Formic acid 11,94
6 Acetic acid 13,08

~u~~
20

0-
(~
min
-20-
0,0 5,0 10,0 15,0 20,0
---

Chromatographic Results:
Test Compound: Succinic acid
Theoretical Plates [11m]: 35080
HETP [~m]: 28.5
Asymmetry: 1.12

~' .//
V-NAGEL
Germany & Export: Tel. ++492421 969-0 France: Tel. ++33 88 682 268
Switzerland: Tel. ++41 62388550 USA: Tel. ++1610559-9848
 Filtration· Testing· Chromatography· Bioanalysis
Filtrieren . Testen . Chromatographie . Bioanalytik

NUCLEOGEL ® Sugar 810 H

The adsorbent NUCLEOGEL® Sugar 810 H is a sulfonated polystyrene/divinylbenzene resin in the H+

form. It has been developed for the chromatographic separation of numerous polar organic compounds
like alcohols, sugar alcohols and sugars. Separation of the analytes is based on a combination of diffe-
rent mechanisms, primarily interactions with the metal ions of the stationary phase. Steric exclusion and
hydrophobic interactions are possible due to the polystyrene skeleton of the phase.
The separation properties of NUCLEOGEL ® Sugar 81 0 H columns are due to a polystyrene resin of the
gel type. This means a low degree of cross-linking and results in some limitations for use of these co-
lumns.
Please note: it is essential that you familiarize yourself with properties
and conditions for work of these columns prior to use.

For preparation of the eluent only use freshly distilled deionized water and analytical grade reagents.
Especially metal ions can strongly influence the separation properties of the cation exchanger. Organic
components in the eluent can cause swelling of the resin with resulting increase in back pressure and
even destruction of the column packing. For removal of insoluble components the eluent should be filte-
red through a 0.451Jm filter. Carefully degas the eluent prior to use, because dissolved gases can cause
detector problems (spikes) at increased temperatures.
The eluent for NUCLEOGEL® Sugar 810 H columns is diluted sulphuric or phosphoric acid (pH 1 - 3).
As organic modifier, you may use up to 30% acetonitrile. When changing to eluents containing acetoni-
trile first flush the column with a 5% acetonitrile mixture at 0.1 - 0.2 mVmin (at least 3 hours). Then you
can equilibrate the column with a higher content of acetonitrile, however, again at a low flow rate. Direct
switching to eluents containing higher acetonitrile concentrations can result in an increase in back pres-
sure and destruction of the column packing.

The column temperature influences retention times, column efficiency and viscosity of the eluent. For
NUCLEOGEL ® Sugar 810 H we recommend a temperature between 20 and 65 cC. During heating and
cooling procedures the column should be flushed with 0.1 ml/min. If necessary the column can be cooled
rapidly by placing it into demineralized water. For many separations the selectivity can by clearly influ-
enced by slight temperature changes. For this reason we recommend use of a column oven or thermo-
stating the lab even for room temperature separations.

The back pressure of NUCLEOGEL ® Sugar 810 H must not exceed 100 bar. Higher pressures result in
a compression of the gel bed and a decrease of separation efficiency. Changes of the flow rate should
be performed in small steps. The maximum flow rate is 1 ml/min. Lower flow rates improve the column
efficiency, however this increases the analysis time. Typical flow rates are between 0.1 and 1 ml/min.
The eluent in the column is 0.01 N sulphuric acid. For conditioning of the column the chromatographic
system is flushed with this eluent and the column is installed, observing the flow direction indicated on
the column. The column is then equilibrated with 20 ml of the eluent using a low flow rate (0.2 mVmin)
and then changed to the eluent of choice.

Polymer-based adsorbents usually show a long life time. However, irreversible adsorptions or a pressure
increase can deteriorate the column effiency. A regeneration or cleaning can in many cases restore the
column performance. For this purpose it is, however, important to analyse the sources of the problem
before using the column for further separations.

Observation possible cause method of regeneration

pressure increase plugging of the filter replace filter elements

elements· .

dead volume see below

compression of incompatible eluent dismount column and allow the gel bed to relax
the gel bed (dead (30 min)
volume) sample not compati-
install the column in the reversed flow direction
ble with the eluent
flush with 0.025 M sulphuric acid at 0.1 ml/min and
heavy contamination 65°C
with metal ions check the column in the normal flow direction

pressure deviations

flow rate too high

working pressure too

high

changes of selec- contamination with install the column in the reversed flow direction
tivity metal ions flush with 0.025 M sulphuric acid at 0.1 mVmin and
65°C
check the column in the normal flow direction

adsorption of hydro- install the column in the reversed flow direction

phobic components flush with 0.025 M sulphuric acid at 0.1 ml/min and
65°C
check the column in the normal flow direction

For storage equilibrate the column with 0.005 M sulphuric acid at a low flow rate. Then carefully close
both ends to avoid desiccation of the column ends. Storage at 4 °C is recommend to avoid microbial
growth.
-1~I--------------M-ACHE9-NAGEl
HPLC columns from MN are quality products especially developed for HPLC analysis. If carefully and properly used excellen
chromatographic results and long column lifetime can be achieved. HPLC columns are designed for qualitative and quantitative analysis c
mixtures of substances and single components.
HPLC columns exclusively must be used in accordance with universally accepted laboratory regulations and HPLC working methods. II
case of doubt about these basic principles, please consult our technical support staff, or your local sales representative.
Before running the column the entire analytical system (column and equipment) must be carefully checked by the operata
Chromatographic conditions (mobile phase, flow, temperature, etc.) must be adapted to the analytical problem. MN does not give an
warranty and is not liable for the success of a separation or application.
We recommend controlling column back pressure regularly. The maximum back pressure should not exceed the limits depicted in the table below.

Maximum pressure [bar]

Inner diameter [mm] 2 3 4 4.6 7.7 8 10 16 21 32 40 50

NUCLEODUR@ 1.8 ~m 900 800 600 600
NUCLEODUR@ 600 600 600 600 400 400 400 400 400 400 400
NUCLEOSIL@50A, 100 A, 120 A 400 400 400 400 400 400 400 400 400 400 400
NUCLEOSIL@300 A 300 300 300 300 300 300 300 300 300 300 300
NUCLEOSIL@500 A 250 250 250 250 250 250 250 250 250 250 250
NUCLEOSIL@ 1000 A, 4000 A 200 200 200 200 200 200 200 200 200 200 200

NUCLEODEX 400 400 400 400 400 400 400 400 400 400 400
NUCLEOGEN@ 200 200 200 200 200
NUCLEOCEL 150 150 150 150
NUCLEOGEL@RP 215
NUCLEOGEL@810 SUGAR H 100
NUCLEOGEL@810 SUGAR Ca 70
NUCLEOGEL@ GPC 150

Follow the general safety instructions for the handling of HPLC solvents used as mobile phases (e.g. Acetonitrile, Methanol) and tE
precautions against any kind of injuries or damage to health (e.g. skin and eye protection in case of broken capillaries).
The disposal of used HPLC columns must follow international, national and local environmental protection laws. The use of HPLC column:
only permitted to staff members, who are qualified in their field. Keep HPLC columns away from children.
MN disclaims and excludes all warranties of any kind or nature whatsoever and MN shall not be liable for any damages (whether dirE
indirect, foreseeable, incidental, compensatory, consequential or special), whether based upon warranty, contract, tort or strict liabilit)
damages and/or losses occur caused by improper use, maintenance, neglect or improper treatment (esp. dismantling of column endfittings ,
opening).

For applicative support

please ask for our
HPLC-Application Guide
or visit our homepage
with more than 3000 l~.l I.
chromatography III I

applications: Reversed Phase HPLC

Application Guide

V-NAGEL
MACHEREY-NAGEL GMBH & CO. KG . Neumann-Neander-Str. 6-8 0-52355 Duren Germany
Germany Switzerland: France: USA:
and international: MACHEREY-NAGEL AG MACHEREY-NAGEL EURL MACHEREY-NAGEL tnc.
Tel.: +49 (0) 24 219690 Tel.: +41 (0) 62 388 55 00 Tel.: +33 (0) 3 88 68 22 68 Tel.: +14848210984
Fax: +49 (0) 24 21969199 Fax: +41 (0) 62 388 55 05 Fax: +33 (0) 3 88 517688 Fax: +14848211272
e-mail: sales-de@mn-net.com e-mail: sates-ch@mn-net.com e-mail: sales-fr@mn-net.com e-mail: sales-us@mn-net.com