5 Chromatographic Assays Employed in the Study
of Alkaliphiles
Phenotypic characterization, especially for anaerobic alkaliphiles, often requires
assays of some fermentative or anaerobic respiration products. Gaseous substances
(e.g., Н 2 , N 2 , СН 4 ) can be analyzed by gas chromatography which is widely used for
their quantification. However, for quantification of soluble substances (sugars,
organic acids, inorganic anions, etc.), precautions should be taken into account, if
chromatographic methods planned to be used. The main source of problems in this
case is the high sodium content (carbonates and chloride) of the medium. For some
haloalkaliphiles, upper limit for growth may reach up to 4 M of sodium. Out of
variety of chromatographic methods, the only insensitive one, working regardless of
the salt content, is a gas-liquid chromatography (GLC). In fact, in order to maintain
the column in good condition, it is sufficient to simply change a glass wool tampon
in a pre-column (sometimes called a liner) equipped with any gas chromatograph.
When an injected sample is evaporated there, salts will stay inside the pre-column
and will not interfere with the column. Although manufacturers recommend change
the tampon time to time, it has become especially topical in case of frequent
injections of the samples with the high salt content. Otherwise, bad resolution and
residual retention of the analyte are possible. With regard to specific protocols of
separation, the manufacturers have their own columns for the separation of acidic,
neutral, or basic substances, and the reader is referred to their catalogs or websites.
The sample preparation for the GLC determination generally includes centrifugation or filtration through membrane filter with a pore size of 0.45 μm. When using
GLC, clarifying the sample is needed not only for the instrument but also for
protecting the needle of the microliter syringe, which is easily clogged in the presence
of insoluble impurities in the sample. After clarification of the sample, next actions
depend on if acidic (e.g., organic acids) or basic (e.g., methylamines) volatile
products are planned to be assayed. To organic acids, a clarified culture medium
should be acidified by formic acid to pH 2–3 to convert nonvolatile organic anions
into volatile free acids. Formic acid is the strongest volatile organic acid and compatible with all types of the columns designed for organic acid separation. Moreover,
it does not detect by the FID detector and thus is “invisible” for the system. Injection
of 1% formic acid into the column before and after a series of analyses is a good
practice for maintaining the column in good condition. If the sample has high buffer
capacity, acidity of formic acid may not be sufficient to shift a pH value into acidic
range. In this case, first, the sample should be acidified by concentrated phosphoric
acid to neutrality and then by formic acid to pH 2–3. Neither hydrochloric nor sulfuric
acid should be used for neutralization as its vapors are highly aggressive toward the
stationary phase of the column. To assay for volatile amines, ammonia solution
should be added to the sample to facilitate amines’ evaporation. Similar to formic
acid, ammonia is also invisible for the FID detector. Interestingly, despite the fact that
the capillary columns almost displaced the packed ones, our experience had shown
that the Supelco packing (0.3% Carbowax 20M + 0.1% H 3 PO 4 , matrix 60/80
74
V. V. Kevbrin
of Alkaliphiles
Phenotypic characterization, especially for anaerobic alkaliphiles, often requires
assays of some fermentative or anaerobic respiration products. Gaseous substances
(e.g., Н 2 , N 2 , СН 4 ) can be analyzed by gas chromatography which is widely used for
their quantification. However, for quantification of soluble substances (sugars,
organic acids, inorganic anions, etc.), precautions should be taken into account, if
chromatographic methods planned to be used. The main source of problems in this
case is the high sodium content (carbonates and chloride) of the medium. For some
haloalkaliphiles, upper limit for growth may reach up to 4 M of sodium. Out of
variety of chromatographic methods, the only insensitive one, working regardless of
the salt content, is a gas-liquid chromatography (GLC). In fact, in order to maintain
the column in good condition, it is sufficient to simply change a glass wool tampon
in a pre-column (sometimes called a liner) equipped with any gas chromatograph.
When an injected sample is evaporated there, salts will stay inside the pre-column
and will not interfere with the column. Although manufacturers recommend change
the tampon time to time, it has become especially topical in case of frequent
injections of the samples with the high salt content. Otherwise, bad resolution and
residual retention of the analyte are possible. With regard to specific protocols of
separation, the manufacturers have their own columns for the separation of acidic,
neutral, or basic substances, and the reader is referred to their catalogs or websites.
The sample preparation for the GLC determination generally includes centrifugation or filtration through membrane filter with a pore size of 0.45 μm. When using
GLC, clarifying the sample is needed not only for the instrument but also for
protecting the needle of the microliter syringe, which is easily clogged in the presence
of insoluble impurities in the sample. After clarification of the sample, next actions
depend on if acidic (e.g., organic acids) or basic (e.g., methylamines) volatile
products are planned to be assayed. To organic acids, a clarified culture medium
should be acidified by formic acid to pH 2–3 to convert nonvolatile organic anions
into volatile free acids. Formic acid is the strongest volatile organic acid and compatible with all types of the columns designed for organic acid separation. Moreover,
it does not detect by the FID detector and thus is “invisible” for the system. Injection
of 1% formic acid into the column before and after a series of analyses is a good
practice for maintaining the column in good condition. If the sample has high buffer
capacity, acidity of formic acid may not be sufficient to shift a pH value into acidic
range. In this case, first, the sample should be acidified by concentrated phosphoric
acid to neutrality and then by formic acid to pH 2–3. Neither hydrochloric nor sulfuric
acid should be used for neutralization as its vapors are highly aggressive toward the
stationary phase of the column. To assay for volatile amines, ammonia solution
should be added to the sample to facilitate amines’ evaporation. Similar to formic
acid, ammonia is also invisible for the FID detector. Interestingly, despite the fact that
the capillary columns almost displaced the packed ones, our experience had shown
that the Supelco packing (0.3% Carbowax 20M + 0.1% H 3 PO 4 , matrix 60/80
74
V. V. Kevbrin