Biotech 1

1. The word "chromatography" derives from the Greek "khròmatos" (color) and
"graphia" (from gràpho "to write"), so it literally means "written in color". Its
origins date back to the early 20th Century (1901-1903), when Russian botanist
Michail Semenovich Tswett filtered a solution of vegetable substances on a col-
umn filled with calcium carbonate, as an adsorbent, and obtained a separation of
the colored pigments. Since then chromatography, in its different applications, has
undoubtedly proven itself as one of the most effective and widely adopted techniques for
qualitatively as well as quantitatively decomposing a mixture of components.
All chromatographic techniques are based on the fact that every compound present in a mixture
interacts differently with the surrounding environment under the same conditions.
Each separation system is determined by the competitive distribution of a component over a mobile
phase (vector fluid or eluent) and a stationary phase. Based on certain parameters, the various sub-
stances migrate differently through the support and thus become separated.
Types of chromatography
Chromatographic techniques vary depending on the physical characteristics of the solute, eluent and stationary
phase which are used to achieve the separation. Overall, five types of chromatography can be identified:
1 Adsorption chromatography (L.S.C.)
This technique is based on the adsorption of a solute on a polar adsorbent such as silica gel, alumina,
diatomaceous earth, magnesium oxide and activated carbon or other compounds that have the same physical
properties. The mechanism governing this kind of separation is the competition of the solute among the polar
groups present on the surface of the stationary phase and the non-polar mobile phase.
2 Partition chromatography (L.L.C.)
The principle behind this technique is based on the separation of a solute between two immiscible solvents,
one of which is the "stationary phase" while the other is the "mobile phase".
Partition chromatography can be divided into:
DIRECT OR NORMAL PHASE CHROMATOGRAPHY - if the stationary phase is more polar than the mobile phase;
REVERSED PHASE CHROMATOGRAPHY - if the mobile phase is more polar than the stationary phase.
3 Ion exchange chromatography
In ion exchange chromatography the surface area of the stationary phase is covered with SO3- / COO- /
NH3+ ionic groups which can be exchanged with the ionic components of the solute. This exchange can be
cationic or anionic.
In this case, the elution depends on the pH of the mobile phase.
4 Exclusion chromatography (GPC)
Exclusion chromatography does not depend on the type of interaction between solute, stationary phase and
mobile phase, but only on the size of the solute molecules in relation to the size of the stationary phase pores
which act as a screen.
Two components can be separated if their molecular weights differ by about 10%.
5 Affinity chromatography
Affinity chromatography is a newly applied method based on specific biochemical interactions between appro-
priately modified stationary phases and molecules of the solute with special spherical or charge characteris-
tics.
CHROMATOGRAPHY
34

2. Chromatographic techniques vary depending on:
a. Physical state of the mobile phase
- chromatography with liquid eluent or liquid chro-
matography (LC);
- chromatography with gas eluent or gas chro-
matography (GC).
In liquid chromatography the elution of a mixture may
be performed in two ways:
- Isocratic elution, using a single eluent and
keeping the flow constant;
- Gradient elution, using two or more eluents
mixed in percentages over time, stepwise, keep-
ing the flow constant.
In gas chromatography, on the other hand, the parti-
tion of a solute in gaseous or vapor phase is transport-
ed by an inert gas through a packed or capillary col-
umn. The gaseous phase, with an appropriately adjust-
ed speed, transports the substance to be analyzed (in
gaseous form) through the stationary phase. Since the
strength with which the stationary phase tends to
retain different components varies, the distinct compo-
nents of the sample will pass though the column at dif-
ferent speeds and exit separately.
b. Presentation of the stationary phase
The solid phase, which may be an active stationary
phase or simply a support, is contained in a column,
and the column is defined as a:
- Packed column
if the column is filled with a finely subdivided solid
such as chromatographic silica sand, brick dust or
glass spheres, homogeneously covered with a liq-
uid;
- Capillary column (or open-tubular column)
if its interior walls are coated with the liquid. These
columns are generally made of fused silica, with a
diameter between 0.2 and 0.6mm and a length
varying from 15 to 50 meters. Compared to packed
columns, capillary columns offer higher resolution
but require longer analysis times.
- Support-coated open-tubular (SCOT) column
is treated so as to form or deposit a porous or
irregular layer on the surface before it is covered
with the liquid phase.
Other chromatographic techniques to mention are:
Headspace gas chromatography (HSGC)
This is an indirect chromatographic technique for quick
determination of volatile compounds in liquids and
solids, in which the instrument parameters must be set
to keep the sample container temperature constant.
The operating principle of this technique is based on
the chromatographic analysis of the vapor phase in
thermodynamic equilibrium above the sample enclosed
in a sealed container.
Under these conditions, the quantity of volatile compo-
nents present in the gaseous phase above the sample
(in the headspace) is higher but proportional to their
concentration in the sample.
Thus, through appropriate calibration, the concentra-
tion of volatile components in solution can be deter-
mined by analyzing a part of the gaseous phase.
Thin-layer chromatography (TLC)
This technique uses a liquid mobile phase and a solid
stationary phase spread in a thin layer on a support,
usually a glass plate.
Used as an alternative to paper chromatography, it
offers:
- higher separation power;
- improved sensitivity;
- shorter analysis times: some separations which
take several hours on paper can be carried out in
just a few minutes, given the appropriate layer.
At the current state of the art, it can reasonably be
assumed that thin-layer chromatography will largely
replace paper chromatography for the separation of
lipophilic substances, but such a replacement seems
premature for the separation of hydrophilic substances.
High-pressure liquid chromatography (HPLC)
In this technique a high-pressure mobile phase is
pumped through a stationary phase made of pressure-
packed microparticle material covered, in some cases,
with a partition liquid.
The advantages offered by this technique, when it can
be applied as an alternative to those previously
described, are as considerable as they are obvious:
- much faster separation and very brief analysis time
(10 - 30 minutes);
- high versatility and the possibility to separate heat-
sensitive and explosive substances, since the
analysis can be conducted at room temperature;
- requires very small sample volumes given the high
sensitivity achieved in UV (nanograms) and fluores-
cence or with electrochemical detectors
(picograms);
- possibility to quickly separate even complex mix-
tures with precision, accuracy and repeatability and
with minimum dexterity skills given the ease of the
process;
- low cost per analysis;
- satisfactory qualitative and quantitative results;
- chromatograms comparable with those obtained
through conventional gravity-induced mobile phase
flow chromatography or through gas chromatogra-
phy, to which this technique is rather similar.
Chromatography
35

3. Adsorbents
Besides the widely used silica gel, other products with particular characteristics are also available and offer a
series of valid alternatives for resolving numerous separation problems.
36
Chromatography
Product Applications Characteristics Size Code
Activated carbon General Powder 250 g 434455
1000 g 434454
Aluminum oxide, acidic Dicarboxylic amino acids, Brockmann 250 g 417185
peptides, etc. 1000 g 417182
Aluminum oxide, basic Polar compounds Brockmann 100 g 417214
1000 g 417217
Aluminum oxide, neutral Ketosteroids, glucoside ketals, Brockmann 250 g 417245
lactones, esters 1000 g 417241
2500 g 417248
Calcium hydroxide General Powder 250 g 433905
Cellulose General Powder 250 g 436061
Clarcel FLO M Solid-liquid separation Cake density: 170-215 g/l 1000 g P8770017
Permeability: 1.4 - 3.8 darcies 5000 g P8770027
Diatomaceous earth compound General Powder 250 g 449895
1000 g 449897
Florisil Column separation of organic 60-100 mesh 100 g 452331
substances 500 g 452333
Pesticide analysis 60-100 mesh 100 g 452271
500 g 452273
Florisil Column separation of organic 100-200 mesh 100 g 452351
substances 500 g 452353
Magnesium oxide General Powder 1000 g 459617
Polyamide TLC Powder 250 g 470021
Purified sand Flash chromatography 40-100 mesh 1000 g 477153
Silica gel TLC Stahl 250 g 453345
1000 g 453347
Flash chromatography 40-63 m 100 g 453351
500 g 453352
1000 g 453353
5000 g 453355
General with wide versatility 60-200 m 500 g 453336
1000 g 453337
5000 g 453332
Silica gel 100 Å General with wide versatility 5 m 1000 g P205RS17
10 m 1000 g P206RS17
16 m 1000 g P207RS17
30 m 1000 g P208RS17
50 m 1000 g P209RS17
Silica gel 300 Å General with wide versatility 5 m 1000 g P210RS17
10 m 1000 g P211RS17
15 m 1000 g P212RS17
20 m 1000 g P213RS17
40 m 1000 g P214RS17
Silica gel 60 Å General with wide versatility 6-35 m 1000 g P2010017
5000 g P2010027
25 kg P2010044
40-63 m 1000 g P2050017
5000 g P2050044
25 kg P2050027
20-45 m 1000 g P2200017
5000 g P2200027
25 kg P2200044
35-70 m 1000 g P2000017
2000 g P2000026
5000 g P2000027
25 kg P2000044
70-200 m 1000 g P2100017
2000 g P2100026
5000 g P2100027
25 kg P2100044
Silica gel 70 Å General with wide versatility 5 m 1000 g P200RS17
10 m 1000 g P201RS17
15 m 1000 g P202RS17
20 m 1000 g P203RS17
40 m 1000 g P204RS17
Silica gel 1000 Å General with wide versatility 5 m 1000 g P215RS17
10 m 1000 g P216RS17
15 m 1000 g P217RS17
20 m 1000 g P218RS17
40 m 1000 g P219RS17

4. Clarcel FLO M is an effective filtration product obtained from expanded raw perlite. It represents an important
alternative to Celite 545, the composition of which causes the user to be exposed to crystalline silica, classified as
a carcinogen by the International Agency for Research on Cancer (IARC).
Its particularly high porosity and low compressibility make this product suitable for solid-liquid separations, with
retention properties up to microscopic dimensions.
It also has the following advantages:
- Improved safety for the users (respirable fraction of the crystalline silica concentration < 1%);
- Insoluble in organic acids and minerals at all temperatures (except hydrofluoric acid);
- Effective even with highly viscous liquids;
- Suitable for a wide variety of filtration conditions, even under vacuum or with high flow rates;
- It gives no odor or color to the separates.
TLC plates
Carlo Erba Reagenti offers a wide range of TLC plates with supports in glass, polyester and aluminum, available in
various formats to meet all the users's needs.
Chromatography
37
Type Thickness Dimensions Quantity Code
(mm) (cm) per box
Analysis plates Polyester support
Alumina 60A F254 0.2 5 x 20 50 PCCM331325
0.2 20 x 20 25 PCCM331254
Alumina 60A 0.2 20 x 20 50 PCCM330254
Silica gel 60 F254 0.25 20 x 20 25 PCCM321354
0.25 5 x 20 50 PCCM321321
Silica gel 60 0.25 20 x 20 25 PCCM320354
0.25 5 x 20 50 PCCM320325
Aluminum support
Silica RP18 F254 0.2 20 x 20 25 PCCM251554
0.2 5 x 20 50 PCCM251525
Silica gel 60 F254 0.2 20 x 20 25 PCCM221254
0.2 5 x 20 50 PCCM221225
Silica gel 60 0.2 20 x 20 25 PCCM220254
Silica gel 60 with indicator 0.2 4 x 8 50 PCCM221205
Alumina 60A F254 20 x 20 25 PCCM231254
Glass support
Silica RP18 F254 1 20 x 20 15 PCCM151653
0.25 20 x 20 25 PCCM151351
0.25 10 x 10 25 PCCM151334
Alumina 60A F254 0.25 20 x 20 25 PCCM131354
Alumina 60A 0.25 20 x 20 25 PCCM130354
0.25 5 x 20 100 PCCM130327
Silica gel 60 F254 0.25 20 x 20 25 PCCM121354
0.25 10 x 20 50 PCCM121345
0.25 10 x 10 25 PCCM121335
0.25 5 x 20 100 PCCM121327
0.25 5 x10 50 PCCM121315
Silica gel 60 0.25 20 x 20 25 PCCM120354
Silica gel 60 0.25 10 x 20 50 PCCM120345
Silica gel 60 0.25 5 x 20 100 PCCM120327
Silica gel 60 0.25 5 x10 50 PCCM120315
Preparative plates Glass support
Alumina 60A F254 1 20 x 20 15 PCCM131653
Silica gel 60 2 20 x 20 12 PCCM120758
1 20 x 20 12 PCCM120653
0.5 20 x 20 25 PCCM120459
Silica gel 60 F254 0.2 20 x 20 25 PCCM121758
variable 20 x 20 25 PCCM121854
1 20 x 20 25 PCCM121653
0.5 20 x 20 25 PCCM121459
HPTLC plates Glass support
Silica 60/10 F254 0.2 10 x 10 25 PCCM111234
0.5 10 x 10 25 PCCM111231
Chiral plates Chiral F254 20 x 20 25 PCCM171354

5. TLC plate visualization systems
TLC visualization is greatly simplified when plates with fluorescence indicators can be used. When placed under a
UV lamp at 254 and 366nm, the substances become visible as dark spots on a fluorescent background.
Alternatively, general reagents can be used, such as iodine, sulfuric acid, acid/nitric acid, sulfuric acid/potassium
dichromate which allow, after appropriate treatment, visualization of the separated products. Some classes of
compounds may in fact be visualized by means of specific reactions, using appropriate chromogenic solutions
sprayed on the TLC plates.
For information on accessories, instruments, columns and HPLC/GC syringes, see our "Labware" catalogue.
Applications
Specific reagents for the preparation of chromogenic solutions.
Solvents
From a qualitative point of view, HPLC solvents have had to adapt to the increasingly specific needs of both
instruments and methods, especially in terms of active impurities which could compromise the correctness and
selectivity of the result by interacting with the analytical complex.
Consequently, our HPLC solvents are accurately and specifically produced by means of forced distillation / filtra-
tion, and then controlled and packaged to adequately meet the needs of this analytical technique.
Solvents for HPLC - LC/MS
Liquid chromatography coupled with mass spectrometry is a recently developed technique which is suitable for
the analysis of fairly polar, non-volatile and thermally stable compounds. It allows the separation and characteri-
zation of many more compounds than GC-MS chromatography and also provides information on the molecular
weight and structure of the HPLC peaks. The significant advantages of this combined technique has lead to its
widespread use in many analytical fields, particularly in the pharmaceutical, environmental and biotechnological
sectors.38
Chromatography
Compound classes Chromogenic solutions
Steroids and sterols, phenols, lipids and sulfides
(blue spots on a yellow background)
Phosphomolybdic acid
Carbohydrates (various shades of blue) Anisic aldehyde (in sulfuric acid, acetic acid and ethanol)
Reducing sugars (various colors) Aniline phthalate (aniline and italic acid in n-BuOH /H2O)
Vitamins A, D, E and terpenes Antimony trichloride, saturated solution in chloroform
Steroids, steroidic glucosides, aliphatic lipids, vitamin A, etc.
(various colors)
Antimony trichloride, saturated solution in chloroform
Halides Silver nitrate / ammonia – acidic fluorescein
A) 1% Ammoniacal AgN03
B) 0.1% Fluorescein in ethanol
Primary amines derived from indole Dimethylaminobenzaldehyde (hydroalcoholic solution)
Aldehydes and ketones (varying from yellow to red) 2,4-Dinitrophenylhydrazine (0.5% in HCI2N)
Phenols (various colors) Ferric chloride (1% in water)
Unsaturated compounds (yellow spots on a pink background) Fluorescein-bromine (0.04% in H2O + Br2 vapors)
Inorganic cations (various colors) 8-Oxyquinoline-Ammonia (NH3 vapors + 0.5 %
oxyquinoline in ethanol - water 60/40)
Barbiturics (gray spots on a colorless background), the same reaction
also occurs for many amines
Mercurous nitrate
Amino acids, aminophosphatides, amino sugars (blue) Ninhydrin (0.2 % in 5% aqueous acetic acid solution)
Halides, except fluorides, dicarboxylic acids
(yellow spots on a purple background)
Bromocresol purple (0.1% in ethanol and ammonia)
Alkaloids, organic bases in general (orange) Dragendorff reagent
A) 1.7% basic bismuth nitrate in 20% acetic acid
B) 40g of potassium iodide in 100ml of H2O
Carboxylic acids (yellow spots on a green background) Bromocresol green 0.3% in methanol / H2O ratio 8:2 -
alkalinized
Product Package Code
Anisic aldehyde 100 ml 415312
Antimony trichloride 100 ml 422834
Basic bismuth nitrate 100 g 428294
Bisublimed iodine 250 g 455955
Bromocresol purple 5 g 470038
Bromocresol purple 0.4% 250 ml 470045
p-Dimethylaminobenzaldehyde 100 g 444604
2,4-Dinitrofluorobenzene 10 g 445701
Fluorescein acid 50 g 452083
Fluorescein sodium 50 g 452113
Hydrindantin 5 g 455291
Product Package Code
Mercurous nitrate 100 g 461154
Ninhydrin 25 g 464922
Nitric acid 69.5 % 1000 ml 408071
8-Oxyquinoline 50 g 467353
Perchloric acid 65% 1000 ml 409111
Phosphomolybdic acid 50 g 405913
Phthalic acid 250 g 406205
Potassium dichromate 500 g 470336
Potassium iodide 250 g 472735
Silver nitrate 25 g 423952
Sulfuric acid 96% 1000 ml 410301

6. LC-MS grade acetonitrile and methanol are available, characterized by:
• Guaranteed specifications of a high-performance
LC solvent (high titer, low acidity, alkalinity and
residue, ideal fluorescence, absorbance/transmit-
tance and gradient test);
• Low metal content, in the order of ppb, in order
to prevent interactions with ionized species in
the mass analyzer;
• Low particulate;
• LC-MS suitability: no signal is greater than the molecular peak of reserpine (609 amu) at the concentration
of 100 ppb, in a range from 50 to 2000 amu.
Solvents for HPLC - Ultragradient grade
The solvents of this product line guarantee excellent short-wavelength performance and limited drift, which
makes them ideal for gradient and trace analysis.
Chromatography
39
Product Size Code
Acetonitrile RS for HPLC - LC/MS 1000 ml 412341
2500 ml 412342
Methanol RS for HPLC - LC/MS 1000 ml 414831
2500 ml 414832
Acetonitrile Methanol
Application HPLC LC-MS HPLC LC-MS
Code 412342 414832
Titer (GC) >= 99,9 % >= 99,9 %
Water <= 0,01 % <= 0,02 %
Non-volatile residue <= 2 ppm <= 2 ppm
Acidity <= 0,0005 meq/g <= 0,0003 meq/g
Basicity <= 0,0002 meq/g <= 0,00006 meq/g
UV transmittance (1 cm - Ref: water)
at 195 nm >= 80 %
at 200 nm >= 93 %
at 210 nm >= 30 %
at 220 nm >= 98 %
at 225 nm >= 65 %
starting from 230 nm >= 98.5 %
at 235 nm >= 85 %
at 250 nm >= 95 %
starting from 260 nm >= 98 %
UV cut off <= 190 nm
Fluorescence (quinine)
at 254 nm <= 1 ppb <= 1 ppb
at 365 nm <= 0.5 ppb <= 1 ppb
HPLC capabilities Binary gradient test H20/CH3CN from 5 Binary gradient test H20/CH30H from 5
to 90% in acetonitrile in 16 minutes at to 90% in methanol in 16 minutes at
1.8 ml/min 1.8 ml/min
Gradient test conforms conforms
at 210 nm <= 1 mA.U.
at 235 nm <= 2 mA.U.
at 254 nm <= 0.5 mA.U. <= 1 mA.U.
LC/MS capabilities TIC 50-2000 m/z ESI(+)
Sensitive impurities (Ref: reserpine) <=100 ppb <= 100 ppb
Metalli
Aluminum (Al) <= 50 ppb <= 50 ppb
Calcium (Ca) <= 50 ppb <= 50 ppb
Iron (Fe) <= 50 ppb <= 50 ppb
Potassium (K) <= 50 ppb <= 50 ppb
Magnesium (Mg) <= 50 ppb <= 50 ppb
Sodium (Na) <= 50 ppb <= 50 ppb
Product Size Code
Acetonitrile Gold ml 1000 412371
ml 2500 412372
ml 4000 412374
ml 5000 412375
Methanol Gold ml 1000 412721
ml 2500 412722
ml 4000 412724
ml 5000 412725
Product Size Code
Acetonitrile Ultragradient Grade mL 1000 P00637U16
mL 2500 P00637U21

7. Solvents for HPLC - Gradient grade
These solvents are high-purity products for very sophisticated uses, with guaranteed fluorescence and transmit-
tance limits.
Mobile phases for HPLC
Clients may request quotes on and order eluent mixtures for HPLC gradient analysis, which provides the following
advantages: time savings, improved certainty and guaranteed quality.
Just send your request to the e-mail address chemicals@carloerbareagenti.com, specifying the solvents of inter-
est, the ratio between their concentrations and the volume requested.
Solvents for HPLC - Isocratic grade
This is a line of solvents which, due to their high purity and strictly controlled chemical-physical parameters, ade-
quately meet the needs of modern analytical HPLC.
40
Chromatography
Product Size Code
Acetonitrile Plus ml 1000 412391
ml 2500 412392
Ethanol Plus ml 1000 412701
ml 2500 412702
Isopropanol Plus ml 1000 412711
ml 2500 412712
Methanol Plus ml 1000 412381
ml 2500 412383
Water Plus ml 1000 412141
ml 2500 412142
Product Size Code
Acetonitrile Gradient ml 1000 P00637G16
ml 2500 P00637G21
ml 5000 P00637G31
Methanol Gradient ml 1000 P09337G16
ml 2500 P09337G21
Product Size Code
Acetone ml 2500 412502
Acetonitrile ml 1000 412411
ml 2500 412412
Anhydrous Ethanol ml 2500 412522
Isopropanol ml 1000 412421
Cyclohexane ml 1000 412431
Chloroform stab. in Ethanol ml 1000 412652
ml 2500 412653
Dichloromethane stab. in Amylene ml 1000 412621
ml 2500 412622
Ethyl Acetate ml 2500 412612
n-Heptane ml 2500 412592
n-Hexane ml 1000 412601
ml 2500 412602
Isooctane ml 1000 412441
Methanol ml 1000 412533
ml 2500 412532
n-Propanol ml 2500 412542
Tetrahydrofuran ml 1000 412451
ml 2500 412452
Toluene ml 2500 412642
Product Size Code
1,2-Dichloroethane ml 1000 P0283716
ml 2500 P0283721
1,4-Dioxane ml 1000 P0363716
1,4-Dioxane stab. in BHT ml 1000 P03637S16
1-Chlorobutane ml 1000 P0223716
2-Methyltetrahydrofuran ml 100 P9963716
ml 2500 P9963721
Acetone ml 1000 P0053716
ml 2500 P0053721
Acetonitrile ml 1000 P0063716
ml 2500 P0063721
Chloroform stab. in Amylene ml 1000 P02437A16
ml 2500 P02437A21
Chloroform stab. in Ethanol ml 1000 P02437E16
ml 2500 P02437E21
Cyclohexane ml 1000 P0253716
ml 2500 P0253721
Dichloromethane stab. in Amylene ml 1000 P02937A16
ml 2500 P02937A21
Dichloromethane stab. in Ethanol ml 1000 P02937E16
ml 2500 P02937E21
Diethyl Ether ml 1000 P0443716
ml 2500 P0443721
Diisopropyl Ether ml 2500 P0433721
Ethyl Acetate ml 1000 P0023716
ml 2500 P0023721
Hexane 99% ml 1000 P052373016
ml 2500 P052373021
Isohexane ml 1000 P6263716
ml 2500 P6263721
Isooctane ml 1000 P0633716
ml 2500 P0633721
Isopropanol ml 1000 P0953716
ml 2500 P0953721
Methanol ml 1000 P0933716
ml 2500 P0933721
ml 5000 P0933729
Methyl tert-Butyl Ether ml 1000 P0923716
ml 2500 P0923721
n-Heptane ml 1000 P0503716
ml 2500 P0503721
n-Hexane ml 1000 P0523716
ml 2500 P0523721
Tetrahydrofuran ml 1000 P0703716
ml 2500 P0703721
Tetrahydrofuran, stabilized ml 1000 P07037S16
ml 2500 P07037S21
Toluene ml 1000 P0713716
ml 2500 P0713721
Water ml 1000 P0153716
ml 2500 P0153721

8. Solvents for preparative HPLC
These products are able to guarantee contamination-free separations and purifications.
Derivatizers
Derivatizers are substances that allow the
chemical modification of a molecule in
order to alter its properties and make it
adaptable to a specific analytical proce-
dure. Originally GLC benefited most from
the advantages of derivatization, but sub-
sequently the technique's applications
extended to other types of analysis such
as mass spectrometry, magnetic nuclear
resonance, UV and visible spectrophotome-
try, fluorimetry, electroanalysis and radio-
analysis.
A selection of the most frequently used
derivatizers are listed in the table on the
right.
Reagents for pre- and post-column modification in HPLC
Both pre- and post-column reactions are used in HPLC: on-line
and off-line. The pre-column reaction allows optimization of
selectivity and sensitivity, improves stability, resolution and peak
symmetry, and allows shortening or lengthening of solute reten-
tion times.
The post-column reaction combined with the use of a fluores-
cent or coloring reagent increase the detector's efficiency. The
availability of a wide range of products which can react on the
functional group greatly facilitates spectrophotometric or fluori-
metric separation and detection of products that would other-
wise be difficult to analyze.
Reagents for ion pair chromatography
This type of chromatography can be used to solve common
analytical problems related to ionic or polarized products.
The following reagents are additives for the mobile phase
that allow the separation in reversed-phase HPLC of ionic
or highly polar substances (counter-ion tetraalkylammoni-
um for anionic electrolytes, alkyl or aryl sulfonate for
cationic electrolytes).
Chromatography
41
Product Size Code
Chloroform stab. in Ethanol ml 2500 438641
Dichloromethane stab. in Amylene ml 2500 463281
Ethyl Acetate ml 2500 448211
Isopropanol ml 2500 415112
Methyl tert-Butyl Ether ml 2500 432022
Toluene ml 2500 488531
Product Size Code
Acetonitrile ml 2500 P00637P21
Ethyl acetate ml 2500 P00237P21
N-Hexane ml 2500 P05237P21
Hexane Mixture of Isomers ml 2500 P0520037P21
Tetrahydrofuran ml 2500 P07037P21
Product Size Code
Benzenesulfonyl chloride ml 10 426231
4-Dimethylaminopyridine g 25 444512
Dimethyldichlorosilane ml 100 444771
N,N,-Dimethylformamide dimethyl acetal ml 10 444901
Heptafluorobutyric acid ml 10 405451
Hexamethyldisilane ml 1 447023
ml 25 447024
Hexamethyldisilazane ml 1 446733
ml 25 446731
Hexamethyldisiloxane ml 1 446743
Methyl isothiocyanate g 100 462531
Methyltrichlorosilane ml 250 462964
ml 2500 462968
N-Acetylimidazole g 25 400713
N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) g 1 461832
N-Trimethylsilylacetamide g 25 489951
N-Trimethylsilylimidazole ml 1 489963
N,0-Bis(trimethylsilyl) acetamide (BSA) ml 1 489933
ml 25 489934
N,0-Bis(trimethylsilyl) trifluoroacetamide (BSTFA) ml 1 489563
ml 25 489561
Pentafluoropropionic anhydride ml 1 x 10 422082
Tetracyanoethylene g 10 487091
Trifluoroacetic anhydride ml 25 422221
ml 100 422222
Trimethylchlorosilane ml 1 489923
ml 100 489921
ml 1000 489926
Vegetable oil characterization: Silylating reagent n. 1 ml 1 X 10 478043
Product Size Code
P-Bromophenacyl bromide g 10 430361
2',7'-Dichlorofluorescein g 10 442541
3,5-Dinitrobenzoyl chloride g 25 445361
2,4-Dinitrofluorobenzene g 10 445701
Fluorescein sodium g 5 452113
g 1000 452117
Fluorescein acid g 50 452083
g 500 452087
Hydrindantin g 5 455291
2-Mercaptoethanol ml 10 460691
Methyl isothiocyanate g 100 462531
Ninhydrin g 5 464928
g 25 464922
o-Phthalaldehyde (OPA) g 10 452751
Product Size Code
Cetyltrimethylammonium bromide 50 g 489833
500 g 489831
Dodecylbenzenesulfonic sodium salt 10 g 405351
1-Heptanesulfonic acid sodium salt 25 g 405851
1-Hexanesulfonic acid sodium salt 25 g 405621
1-Octanesulfonic acid sodium salt 25 g 405861
1-Pentanesulfonic acid sodium salt 25 g 405841
Tetramethylammonium acid sulfate 250 g 487101
Tetramethylammonium hydroxide 10% 100 ml 487491
p-toluenesulfonic acid sodium salt 100 g 411504

9. Standards for ion chromatography
Our standard solutions for ion chromatography are obtained by dissolution of a high-purity salt (+99.9%) in
water. They are characterized by:
• Concentrations equal to 1000 ppm;
• Guaranteed titer with a tolerance of 0.2% at the 95% confidence level;
• Raw materials selected and verified against N.I.S.T. Standard Reference Materials;
• Available in 50,100,250 and 500ml HDPE bottles;
• Certificate of analysis with references on the analytical method, the N.I.S.T. Standard Reference Materials
and the confidence interval;
• Shelf life, for the unopened product package, of 2 years.
For instrument calibration, the following multi-ion standard solutions are available for ion chromatography, com-
plete with certificate of analysis with references on the analytical method, the N.I.S.T. Standard Reference
Materials and the confidence interval.
Carlo Erba Reagenti offers its clients the possibility of requesting quotes on and ordering custom-made multi-ion
solutions.
Just send your request to the e-mail address chemicals@carloerbareagenti.com, specifying the ions of interest,
their respective concentrations and the volume requested.
Concentrated mobile phases for ion chromatography
The following eluents are filtered at 0.2µm and prepared from ultra-pure salts and 18-megaohm deionized water.
These are concentrated solutions that should be diluted by a factor of 100.
The shelf life for the unopened product package is 2 years.
42
Chromatography
Code
Ion Matrix Concentration 50 ml 100 ml 250 ml 500 ml
Ammonium (NH4
+) water 1000 ppm 503310 503311 503312 503313
Bromates (BrO3
-) water 1000 ppm 503170 503171 503172 503173
Bromides (Br-) water 1000 ppm 503210 503211 503212 503213
Calcium (Ca2+) water 1000 ppm 503220 503221 503222 503223
Cyanides (CN-) water and sodium hydroxide 1000 ppm - 503358 - -
Chlorates (ClO3
-) water 1000 ppm 503180 503181 503182 503183
Chlorites (ClO2
-) water 1000 ppm 503190 503191 503192 503193
Chlorides (Cl-) water 1000 ppm 503230 503231 503232 503233
Chromates (CrVIO4
2-) water 1000 ppm 503240 503241 503242 503243
Fluorides (F-) water 1000 ppm 503250 503251 503252 503253
Phosphates (PO4
3-) water 1000 ppm 503340 503341 503342 503343
Iodides (I-) water 1000 ppm 503260 503261 503262 503263
Lithium (Li+) water 1000 ppm 503280 503281 503282 503283
Magnesium (Mg2+) water 1000 ppm 503290 503291 503292 503293
Nitrites (NO2
-) water 1000 ppm 503320 503321 503322 503323
Nitrates (NO3
-) water 1000 ppm 503330 503331 503332 503333
Potassium (K+) water 1000 ppm 503270 503271 503272 503273
Sodium (Na+) water 1000 ppm 503300 503301 503302 503303
Sulfates (SO4
2-) water 1000 ppm 503350 503351 503352 503353
Code
Ion Matrix Concentration 100 ml
8 anions: Fluorides (F-), Chlorides (Cl-), Bromides (Br-), Nitrites (NO2
-),
Nitrates (NO3
-), Phosphates (PO4
3-), Sulfates (SO4
2-), Acetates (CH3COO-) acqua 1000 ppm per ciascun ione 504517
Description Matrix Size Code
0.18 M Sodium carbonate / 0.17 M Sodium bicarbonate water 100 ml 504530
0.22 M Sodium carbonate / 0.28 M Sodium bicarbonate water 100 ml 504531
0.35 M Sodium carbonate / 0.1 M Sodium bicarbonate water 100 ml 504532
0.5 M Sodium carbonate water 100 ml 504533
0.17 M Sodium bicarbonate water 100 ml 504534