The choice of the detection is critical in HPLC as only compounds can be analyses if they are detected. Using a not suitable detector for the compounds of interest the chromatographic information to this compound will get lost. To select the most appropriate detection mode, four important parameters should be taken into consideration; chemical nature of the analytes, potential interferences, LOD and LOQ required, linearity range, availability and/ or cost of detector. Below are some of the most common detection techniques for liquid chromatography presented. Fluorescence, electrochemical or mass detectors should be used for trace analysis. For preparative HPLC, refractive index is preferred because it can handle high concentrations without overloading the detector.
Ultraviolet/Visible
Absorbance (UV/Vis)
UV detectors are most commonly used in HPLC. This detector is a robust, inexpensive and versatile detection technique since most compounds absorb light, especially at low UV wavelengths. It is possible to use a diode array detector (DAD) and allow monitoring at multiple wavelengths simultaneously. The downside is that a UV detector is not analyte specific and requires that the analyte absorb more light than sample matrix at the set wavelength.
Choose a detection wavelength that maximizes sensitivity and specificity, but keep in mind that the mobile phase solvents and buffer components may cause slight shifts in UVmax from reference values. Therefore, it is advisable to check the analyte absorbance in the mobile phase. Mobile phase solvents and buffer components also have UV cut-off; therefore, make sure to work well above these levels. Otherwise, there are likely to be problems with reduced sensitivity and increased system noise (unstable and drifting baseline noise). UV wavelengths below 200 nm should be avoided because detector noise increases in this region. Higher wavelengths give greater selectivity.
Refractive index (RI)
Refractive index is also a common detection technique, and measures the difference in the refractive index of a sample cell versus a reference cell. This detector is also a non-selective detection technique, being concentration dependent. The sensitivity is typically 100–1000 times lower than a UV/Vis detector. The benefit over a UV detector is the possibility to quantify analytes with no chromophores in the molecular backbone. The drawback is the sensitivity and the fact that RI detectors are typically used in isocratic mode only.
Fluorescence (FL)
Fluorescence detection is specific and measures only compounds that fluoresce; hence, a requirement of this technique. The operation is similar to a UV/Vis detector but where the detector flow cell is used as the sensor through which excitation light passes axially. A photocell is located at the side of the cell to receive radially emitted light.
The cell wall is made of special glass to prevent the excitation light or other stray light from reaching the photocell. When a solute that fluoresces in the excitation light flows through the cell, the molecule excites and fluorescent light passes through the walls of the cell onto the photocell. The excitation light may be light of any wavelength selected from the light source using a monochrometer.
Another monochrometer may also be used to selectively analyze the fluorescent light and thus, a fluorescent spectrum can be produced for excitation light of any specific wavelength and an excitation spectrum produced for fluorescent light of any specific wavelength.
To improve specificity of an LC analysis, a fluorescent derivatization reagent can be added (either pre-column or post-column) to form a fluorescent derivative of the substance of interest. This derivative may then be selectively detected from other solutes, which, (if they do not fluoresce) need not be resolved from each other by the separation column. Fluorescence detection is up to 1000 times more sensitive than UV/Vis, and is also concentration sensitive.
Evaporative light
scattering (ELS)
ELS is also a non-selective detection technique, but where the ELS detector (ELSD) is mass sensitive and not concentration dependent. It is an ideal technique for high molecular weight compounds, sugars, and less volatile acids. The detector measures the light scattering and where the amount of scattering is related to the molecular mass of the analyte, i.e. the more mass the more scattering will be seen measured. In the detector, there are three processes; nebulization of the mobile phase (1), evaporation of the mobile phase (2) and light scattering by analyte particles. In contrast to RI, it works well in gradient mode. Keep in mind that mobile phase solvents should be volatile for best performance.
Electrochemical (EC)
An electrochemical detector requires that the analytes can be oxidized or reduced by an electrical current. The detector output is an electron flow generated by a reaction that takes place at the surface of electrodes. If this reaction is complete (exhausting all the analyte), the current becomes zero and the generated total charge is proportional to total mass of material that has been reacted. This process is called coulometric detection.
If the mobile phase is continuously flowing past the electrodes, the reacting analyte is continuously replaced in the detector. As long as the analyte is present between the electrodes, a current will be maintained, albeit varying in magnitude, and is called amperometric detection. An electrochemical detector requires three electrodes, the working electrode (where oxidation or reduction takes place), the auxiliary electrode and the reference electrode (compensates for changes in the background conductivity of the mobile phase). Electrochemical detection is more sensitive than fluorescence detection, but commonly not as selective as fluorescence and generally not compatible with gradient elution.
Mass spectrometer
(MS)
Mass spectrometry is regarded as an established, routine, detection technique. MS detectors can be coupled to various separation techniques such as liquid chromatography (LC), thin layer chromatography (TLC), or gas chromatography (GC), where the hyphenation with LC is by far the most frequent setup. In contrast to more simple detectors, i.e. UV, RI, FL etc., MS generates data about molecular masses and detailed structural parameters and thereby offers the possibility to discriminate between co-eluting peaks in selected ion monitoring mode.
The latter reduces the requirement for chromatographic retention and resolution before detection, yet it is always better to have retained and completely resolved peaks to prevent ion suppression or ion enhancement effects. Mass analyzers can be quadrupole, magnetic sector, time-of-flight, ion trap, or ion cyclotron resonance type.
A quadrupole mass analyzer consists of four parallel rods that have fixed direct current (DC) and alternating radio frequency (RF) potentials applied to them. The HPLC system handles dissolved analytes under ambient pressure (760 Torr) and delivers the sample to the MS, where the detection of the gaseous, ionized samples is performed under high vacuum conditions (10-5-10-6 Torr). The transfer of the analyte solution from the LC to the MS is accomplished via an interface. The interface converts the sample stepwise to an aerosol, ionizes it, and removes the solvent. Ions are then focused and passed along the middle of the quadrupoles.
Their movement will depend on the electric fields so that only ions of a particular mass to charge ratio (m/z) will have a stable path to the detector. The RF is varied to bring ions of different m/z into focus on the detector and thus build up a mass spectrum. Depending on the physical properties and the molecular mass of the molecules, different types of interfaces are used, which vary among each other by how they ionize the molecules and the pressure applied during this process.
At present, all the common ionization techniques operate under ambient pressure; i.e. electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), matrix assisted laser desorption/ionization (MALDI), and atmospheric pressure photo ionization (APPI). ESI and APCI are by far the most widely used in LC-MS hyphenation.
The more esoteric techniques, electron ionization (EI) and chemical ionization (CI) work under high vacuum conditions with the advantage of being suitable for GC-MS hyphenation. Quadrupole mass spectrometers commonly have two configurations when used with liquid-chromatography, either as a simple single quadrupole system or placed in tandem. The latter principle, the triple quadrupole mass spectrometer, enables ion fragmentation studies (tandem mass spectrometry or MS/MS) to be performed.
Electrospray
Ionization (ESI)
In ESI mode, liquid solutions of charged or polar substances, delivered with an HPLC system, are sprayed utilizing a metal capillary (“spray needle”) and a nebulizer gas (nitrogen) in the MS. Resulting droplets are dried (desolvatization) and volatilized, isolated, analyte ions are transferred to the detector. Thermal stress is low so the analyte molecules do not decompose. ESI is almost unlimited regarding molecule size and suitable for medium to strong polar molecules, e.g., amines, carboxylic acids, heteroaromatics, and sulfonic acids.
ESI is applied when fragmentations are unwanted and molecular masses of biomolecules have to be determined. ESI-MS is well suited for hyphenation with LC, and as long as flow rates do not exceed maximum 1–2 mL/min (depending on instrumentation), attainable sensitivity is very high; however, flow rates of between 1–500 μL/min are more common. In liquid solution, molecules are either already ionized, or will become protonated or deprotonated by additives in the sample solution and the mobile phase. To achieve best sensitivity, the mobile phases used should be set at a pH where analytes are ionized, and a rule of thumb is to use neutral to basic pH (7–9) for acids, whereas more acidic pH (3–4) is advisable for basic compounds. If the analytes of interest have multiple pKa values and may change their ionization state, other pH values may be more beneficial both in terms of ionization of the analyte and behavior in the column.
Thus, depending on the choice of solvent and additives, either positive and/or negative ESI mode can be used. Typically, positive mode is applied in combination with more basic molecules, while acid compounds are analyzed in negative mode. 0.1% formic acid is commonly added to the mobile phase in positive ESI mode to provide a low pH (≈3) and to protonate the analyte(s). Acidic analytes will be neutralized under such conditions, accordingly negative ESI mode is preferred and higher mobile phase pH is recommended. Volatile buffers like ammonium acetate or ammonium formate are used in the pH range 4.5–7 to deprotonate the analyte(s), and for high pH, it is possible to use either ammonium carbonate or ammonium hydroxide (aqueous ammonia). For both negative and positive ESI, it is a prerequisite that all mobile phase solvents and additives are volatile in order to avoid contamination of the mass spectrometer, and that the total mobile phase ionic strength is adequate (generally 2–25 mM) to prevent unnecessary down-time for cleaning of the detector.
Strong acids like hydrochloric acid or nitric acid are unsuitable for two reasons: they form ion pairs with analyte molecules (analyte signal suppression) and display strong oxidizing properties. Trifluoroacetic acid (TFA) is a special case: It is widely used as an ion-pairing reagent to improve the liquid chromatographic separation of peptides or proteins.
On the other hand, TFA can cause strong ion suppression in mass spectrometry (mainly in negative ESI mode) and contaminates the LC-MS system. ’A good compromise here would be through the use of difluoroacetic acid (DFA). DFA provides the same excellent increase in efficiency as TFA but without as much ion suppression nor does it contaminate the MS system as readily as TFA.
Unfortunately, both a quantitative estimation of these effects as well as general recommendations is not possible as their strength strongly depends on the MS system used. Triethylamine as an alternative additive behaves in a similar manner. If the use of TFA is unavoidable, a weak acid such as propanoic acid, or isopropanol can be added to the mobile phase in order to decrease a signal suppression effect.
Buffers do not only adjust the pH of the eluent and lead to ionization of a target molecule, they can also form adducts with the analyte. Adducts [M + buffer], e.g. with ammonium, alkali, halogens, formate or acetate, will lead to the detection of an additional peak in the MS spectrum; even a complete suppression of the analyte signal is possible when the vapor pressure of the resulting adduct (mainly alkali) is decreased significantly. Due to this, and in order to keep the ESI source clean, volatile buffers are recommended.
Non-volatile salts like phosphates, borates, sulfates or citrates will precipitate in the MS source, block it, and cause tedious cleaning procedures.
Atmospheric pressure
chemical ionization (APCI)
This technique is complementary to ESI and also useful for LC-MS hyphenation. It does not require a mobile phase with conducting properties where acetone or acetic acid esters can be used as solvents and thus allows for a coupling of APCI with normal phase chromatography. In APCI mode, the analyte solution is vaporized prior to the ionization. Subsequently solvent molecules (aqueous-organic, e.g. methanol, propanol, acetonitrile, acetone etc., combined with 2–20 mM of a volatile organic buffer such as formic or acetic acid, ammonium acetate, ammonium formate or triethylamine) become ionized with a corona needle where their charge is then transferred to the analyte molecules via proton transfer or abstraction.
APCI is suitable for the analysis of less polar, weakly ionizable substances with small or medium molecular weight (analytes without acidic or basic functional groups, e.g. hydrocarbons, alcohols, aldehydes, ketones, esters) and is therefore complementary to ESI, as long as the sample is thermally stable and vaporizable.
Fragmentations are generally observed with APCI. Highest sensitivity is achieved using acetonitrile, methanol or water as solvents, and where the degree of analyte ionization can be optimized via mobile phase pH. As for ESI, flow rates up to maximum 1–2 mL/min can be tolerated. There are other less commonly used detection techniques possible to combine with liquid chromatography, such as chemiluminescence nitrogen (CLND), radio detectors, charged aerosol (CA, inductive coupled plasma (ICP), nuclear magnetic resonance (NMR), but these are not dealt with here.
.JPG)
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