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Article-008 (1)
Article-008 (1)
Review Article
concentrations ranging from 2 to 50 mM, although a depositions on the metal surfaces, and could completely
maximum concentration of 10–20 mM is recommended block ion transmission. In addition, if the anion and cation
to avoid ion suppression. The acceptable buffer pair too strongly with the analyte, then the analyte ions
concentration that does not adversely affect ionization might be prevented from carrying the excess charge on
has been observed to be highly compound dependent. A the droplet surface; as a result, the ESI response may be
useful rule of thumb is to use as low a concentration of very low. For those reasons, when ESI is interfaced with
buffers as possible to give reasonable chromatographic HPLC, volatile buffers composed of weak acids and bases
performance5. must replace non-volatile modifiers. Also, strong acids
such as trifluoroacetic (TFA), heptafluorobutyric, and
Criteria for choosing Buffers and concept of volatile and
hydrochloric acid, which are used as ion-pairing agents,
non volatile buffers
tend to mask the analyte signal in ESI-MS. Electron
Buffers should be chosen such that the pH of the mobile ionization, used in Direct-EI, is not influenced by the
phase falls within the buffer’s natural pKa range for presence of preformed ions in the form of salts or strong
maximum buffering capacity. acids and bases so that the analyte can be ionized
independently. Because of the extremely reduced liquid
Buffer capacity outside of these ranges is limited,
intake, Direct-EI slowly displaces a negligible salt
particularly at lower concentrations. N-methyl
deposition within the ion chamber as the only sign of a
morpholine may be used at higher pH ranges and has
salt presence. After many days of continuous operation,
shown utility in improving sensitivity and
the salt deposition is barely visible in the ion source, and
chromatographic peak shape for negative ion APCI
can be removed readily by routine maintenance
analysis6. Ammonium adducts can be observed in positive
procedures before any changes in the performance are
ion mode and formate or acetate adducts in negative
observed.
mode at higher buffer concentrations. Adduct formation
is detrimental only in that it can cause greater variability The ion suppression/enhancement in LC/ESI/MS strongly
and loss of detection sensitivity for the analyte. These depends also on the additives of the mobile phase
adducts can sometimes be destroyed by increasing the employed. Additives and buffers in LC mobile phase are
source temperature, voltage, or both. Formic or acetic primarily employed to achieve reproducible retention
acid concentrations of 0.1–1% (v/v) are recommended time due to their buffering capacity and, secondly, to
when preparing low pH mobile phases to enhance produce sharper peak shape if used as an ion pairing
ionization in electrospray. Trifluoroacetic acid is preferred agent capable of creating neutral species with the target
for protein and peptide separations but should be analyte8.
avoided when negative ion mode is utilized. Ammonium
Additives and Buffers in LC-MS/MS
hydroxide, or, in rare cases, triethylamine, are
recommended for high pH mobile phases. For basic In LC/ESI/MS the additives and buffer can serve to
compounds, 0.1% acid should be mixed with the organic protonate basic molecules when operating in positive ion
component, whereas water or neutral buffer should be mode and vice versa for acidic molecules. Generally in
used for neutral or acidic species. The previously LC/ESI//MS analysis, volatile buffers and additives (i.e.
discussed buffers can be used as the aqueous component ammonium acetate, formate or hydroxide, formic or
to improve peak shape and resolutions by providing acetic acid or trifluoroacetic acid) are preferred to the
greater control of pH. A direct consequence of the non-volatile ones (i.e. phosphates, sulphates, borates and
reduced flow rate is the possibility to introduce a steady citrates). In fact the non-volatile eluent components can
stream of a non-volatile buffer as a mobile-phase be deposited on the ion surface, leading to contamination
modifier. A micro-particle-beam interface before, and of the electrodes inside the ion source. This
Cap-EI later, demonstrated that the combination of a contamination reduces the effective charge density at the
reduced flow rate and electron ionization enhanced the electrode surface, emulating a lower voltage for the
ruggedness and flexibility to tolerate chemically electrode: a reduction in the signal intensity will be
“aggressive” mobile phases7. observed over time. However, volatile buffer and additive
must be used at a concentration as low as possible,
One of the most challenging applications in LC–MS is to
consistently with chromatographic resolution. In fact
enhance the tolerance toward the non-volatile buffers
buffer and additive species compete for surface site on
that are added to the mobile phase to improve
the electrosprayed droplets and an increase in their
chromatographic separation in several applications of
concentration leads to decrease in the analyte signal: the
biological and environmental interest. As a matter of fact,
competition for surface sites of the droplets favours the
the presence of non-volatile, ionic species (phosphate and
higher concentration species and, in this case, analyte
sulphate buffers) in the ESI spray is deleterious. One of
species become depleted. This effect is compound-
the most critical factors in adapting LC methods is the
dependent: some analytes show only a small decrease of
choice of buffer. In volatile buffers interfere with good
signal while for others the response can be decreased
MS performance. For the best long-term performance, it
much more. An interesting study on the ion
is highly recommended that the method be modified to
suppression/enhancement due to additives and the
use a volatile buffer. Non-volatile species cause salt
8. C.R. Mallet, Z. Lu, J.R. Mazzeo, Ion suppression due 10. A Hutchaleelaha, J Sukbuntherng, HH Chow, M
to additives, Rapid Commun. Mass Spectrom, 18, Mayersohn, Practical guidelines of preparing
2004, 49–58. mobile phases in LC-MS/MS, LC/GC, 21(5), 2001,
1840-1858.
9. BA Thomson, Atmospheric pressure ionization and
liquid chromatography/mass spectrometry
together at last, J Am Soc Mass Spectrom, 9, 1998,
187–193.
Mr. Dharmendra Patel graduated at Rajiv Gandhi University of Health Sciences, Karnataka, India
and currently at post graduation level in K.L.E. University, taken specialization in Quality
Assurance. Currently working as a project trainee at Cadila Pharmaceuticals Limited, Ahmedabad,
Gujarat, India.