Carbopack C) provides better separation for all structurally possible C2–C5 acids
than well-known FFAP capillary columns from different manufacturers. Nonvolatile
substances (sugars, organic acids like succinic) can also be analyzed by the GLC
method, but derivatization is required to convert them into volatile form [137] that
complicates the assay.
The HPLC method developed for analyses of both volatile and nonvolatile substances are based on direct injection of the sodium containing sample into the eluent.
Analysis of brines is a heavy test for any chromatographic system, and, until now,
the “universal” column or common solution capable to assay any product or
metabolite in the whole range of the sodium solubility still does not exist. Although
pre-columns, cartridges, and centrifugal mini-columns (mini-inserts) for desalting
the sample are available, they do not absorb sodium entirely at its high content, and
an excess sodium can enter the column leading to unsatisfactory separation. Our
experience with the widely used Aminex HPX-87H (BioRad) column (used to
separate neutral and acidic fermentation products) has shown that the column can
operate effectively up to 0.5–1.0 M of sodium, but above this concentration, the
efficiency significantly deteriorates. Some success can be achieved if the peak of
the target analyte is far enough from the “water (or salt) peak” (retention time for the
void volume of the column). Significant progress has been made by Thermo
Scientific Dionex in their IonPac AS5 column designed for the separation of strongly
retained species such as thiocyanate and thiosulfate in 7% brine. Unfortunately, this
approach will not work for every desired species, and, again, there is no common
solution for every microbiologically interesting substance.
The sample preparation for the HPLC determination requires more attention,
especially when an acid eluent is used. As applied to the Aminex HPX-87H column,
after removing the cells, an alkaline culture fluid should be acidified by sulfuric acid
to pH 2–3. Using hydrochloric acid is not recommended by most manufacturers as it
has a corrosive effect even for the stainless steel. If a highly concentrated carbonate
medium was used, a vigorous foaming is observed so this operation should be
carried out in vials of larger volume to avoid spilling out the sample. The evolved
carbon dioxide remains partly dissolved in the sample, and if the acidified sample is
immediately injected into the column, a negative peak of CO 2 can be seen in the field
of analytes’ peaks when using a refractometric detector. Particularly for the Aminex
HPX-87H column, a CO 2 peak is close to the propionate peak and interferes
quantitatively in the estimation of the latter. To avoid this, the acidified samples
are best left closed before injection for several hours or overnight. Another precaution is associated with the presence of sulfide in the anaerobic media. As pH of the
eluent is acidic, sulfide is converted to hydrogen sulfide which, in turn, is easily
oxidized to insoluble sulfur. Depending on its content, numerous injections of the
sulfide-containing samples can lead to clogging of capillaries and the column. Out of
our experience, if the sample contains no more than 0.5 g/L of Na 2 SÁ9H 2 O (a widely
used concentration of sulfide as a reductant for anaerobic media), there is no need to
take special efforts, and the best solution will be to leave the acidified sample for
overnight for spontaneous oxidation of the sulfide inside the vial. The amount of
elemental sulfur formed is negligible in this case; the sample remains clear and can
Isolation and Cultivation of Alkaliphiles
75
than well-known FFAP capillary columns from different manufacturers. Nonvolatile
substances (sugars, organic acids like succinic) can also be analyzed by the GLC
method, but derivatization is required to convert them into volatile form [137] that
complicates the assay.
The HPLC method developed for analyses of both volatile and nonvolatile substances are based on direct injection of the sodium containing sample into the eluent.
Analysis of brines is a heavy test for any chromatographic system, and, until now,
the “universal” column or common solution capable to assay any product or
metabolite in the whole range of the sodium solubility still does not exist. Although
pre-columns, cartridges, and centrifugal mini-columns (mini-inserts) for desalting
the sample are available, they do not absorb sodium entirely at its high content, and
an excess sodium can enter the column leading to unsatisfactory separation. Our
experience with the widely used Aminex HPX-87H (BioRad) column (used to
separate neutral and acidic fermentation products) has shown that the column can
operate effectively up to 0.5–1.0 M of sodium, but above this concentration, the
efficiency significantly deteriorates. Some success can be achieved if the peak of
the target analyte is far enough from the “water (or salt) peak” (retention time for the
void volume of the column). Significant progress has been made by Thermo
Scientific Dionex in their IonPac AS5 column designed for the separation of strongly
retained species such as thiocyanate and thiosulfate in 7% brine. Unfortunately, this
approach will not work for every desired species, and, again, there is no common
solution for every microbiologically interesting substance.
The sample preparation for the HPLC determination requires more attention,
especially when an acid eluent is used. As applied to the Aminex HPX-87H column,
after removing the cells, an alkaline culture fluid should be acidified by sulfuric acid
to pH 2–3. Using hydrochloric acid is not recommended by most manufacturers as it
has a corrosive effect even for the stainless steel. If a highly concentrated carbonate
medium was used, a vigorous foaming is observed so this operation should be
carried out in vials of larger volume to avoid spilling out the sample. The evolved
carbon dioxide remains partly dissolved in the sample, and if the acidified sample is
immediately injected into the column, a negative peak of CO 2 can be seen in the field
of analytes’ peaks when using a refractometric detector. Particularly for the Aminex
HPX-87H column, a CO 2 peak is close to the propionate peak and interferes
quantitatively in the estimation of the latter. To avoid this, the acidified samples
are best left closed before injection for several hours or overnight. Another precaution is associated with the presence of sulfide in the anaerobic media. As pH of the
eluent is acidic, sulfide is converted to hydrogen sulfide which, in turn, is easily
oxidized to insoluble sulfur. Depending on its content, numerous injections of the
sulfide-containing samples can lead to clogging of capillaries and the column. Out of
our experience, if the sample contains no more than 0.5 g/L of Na 2 SÁ9H 2 O (a widely
used concentration of sulfide as a reductant for anaerobic media), there is no need to
take special efforts, and the best solution will be to leave the acidified sample for
overnight for spontaneous oxidation of the sulfide inside the vial. The amount of
elemental sulfur formed is negligible in this case; the sample remains clear and can
Isolation and Cultivation of Alkaliphiles
75