Bioprocess engg. & technology

msbo309

Experiment 6

Aim of the Experiment

Analytical techniques like HPLC,FPLC,GC,GC-MS etc. For measurement of amounts of products/substrates

Principle

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

It is evident from equations 11.1 to 11.13 that the resolving power of a chromatographic column is determined by a number of factors that are embedded in equation. This shows that the resolution increases with:

• the number of theoretical plates (N) in the column and hence plate height (H). The value of N increases with column length but there are practical limits to the length of a column owing to the problem of peak broadening.

• the selectivity of the column, a; and

• the retentivity of the column as determined by the retention factor, k.

As the number of theoretical plates in the column is related to the surface area of the stationary phase, it follows that the smaller the particle size of the stationary phase, the greater the value of N, i.e. N is inversely proportional to particle size. Unfortunately, the smaller the particle size, the greater is the resistance to the flow of the mobile phase for a given flow rate. This resistance creates a backpressure in the column that is directly proportional to both the flow rate and the column length and inversely proportional to the square of the particle size. The back-pressure may be sufficient to cause the structure of the matrix to collapse, thereby actually further reducing eluent flow and impairing resolution. This problem has been solved by the development of small particle size stationary phases, generally in the region of 510 mm diameter with a narrow range of particle sizes, which can withstand pressures up to 40 MPa. This development, which is the basis of HPLC that was originally and incorrectly referred to as high-pressure liquid chromatography, explains why it has emerged as the most popular, powerful and versatile form of chromatography. Larger particle size phases are available commercially and form the basis of low-pressure liquid chromatography in which flow of the eluent through the column is either gravity-fed or pumped by a low pressure pump, often a peristaltic pump. It is cheaper to run than HPLC but lacks the high resolution that is the characteristic of HPLC. Many commercially available HPLC systems are available and most are microprocessor controlled to allow dedicated, continuous chromatographic separations.

GAS CHROMATOGRAPHY

The principles of gas chromatography (GC) are similar to those of HPLC but the apparatus is significantly different. It exploits differences in the partition coefficients between a stationary liquid phase and a mobile gas phase of the volatilised analytes as they are carried through the column by the mobile gas phase. Its use is therefore confined to analytes that are volatile but thermally stable. The partition coefficients are inversely proportional to the volatility of the analytes so that the most volatile elute first. The temperature of the column is raised to 50-300 oC to facilitate analyte volatilisation. The stationary phase consists of a high-boiling-point liquid material such as silicone grease or wax that is either coated onto the internal wall of the column or supported on an inert granular solid and packed into the column. There is an optimum flow rate of the mobile gas phase for maximum column efficiency (minimum plate height, H). Very high resolutions are obtained hence the technique is very useful for the analysis of complex mixtures. Gas chromatography is widely used for the qualitative and quantitative analysis of a large number of low-polarity compounds because it has high sensitivity, reproducibility and speed of resolution. Analytically, it is a very powerful technique when coupled to mass spectrometry.

Procedure

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Columns

Conventional columns used for HPLC are generally made of stainless steel and are manufactured so that they can withstand pressures of up to 50MPa. The columns are generally 3–25 cm long and approximately 4.6mm internal diameter to give typical flow rates of 13cm3 min–1. Microbore or open tubular columns have an internal diameter of 1–2mm and are generally 2550 cm long. They can sustain flow rates of 520mm3 min–1. Microbore columns have three important advantages over conventional columns:

• reduced eluent consumption due to the slower flow rates;

• ideal for interfacing with a mass spectrometer due to the reduced flow rate; and

• increased sensitivity due to the higher concentration of analytes that can be used.

Matrices and stationary phases

Two main forms of matrix/stationary phase material are available, based on a rigid solid structure. Both forms involve approximately spherical particles of a uniform size to minimise space for diffusion and hence band broadening to occur. They are made of chemically modified silica or styrene/divinylbenzene copolymers. The two forms are:

• Microporous supports: In which micropores ramify through the particles that are generally 510 mm in diameter.

• Bonded phases: In which the stationary phase is chemically bonded onto an inert support such as silica.

Mobile phases

The choice of mobile phase to be used in any separation depends on the type of separation to be achieved. Isocratic elution may be made with a single pump, using a single eluent or two or more eluents premixed in fixed proportions. Gradient elution generally uses separate pumps to deliver two eluents in proportions predetermined by a gradient programmer. All eluents for use in HPLC systems must be specially purified because traces of impurities can affect the column and interfere with the detection system. This is particularly the case if the detection system is based on the measurement of absorbance changes below 200 nm. Pure eluents for use in HPLC systems are available commercially, but even with these a 15mm microfilter is generally introduced into the system prior to the pump. It is also essential that all eluents be degassed before use otherwise gassing (the presence of air bubbles in the eluent) tends to occur in most pumps. Gassing, which tends to be particularly bad for eluents containing aqueous methanol and ethanol, can alter column resolution and interfere with the continuous monitoring of the eluate. Degassing of the eluent may be carried out in several ways – by warming, by stirring vigorously with a magnetic stirrer, by applying a vacuum, by ultrasonication, and by bubbling helium gas through the eluent reservoir.

Pumps

Pumping systems for delivery of the eluent are one of the most important features of HPLC systems. The main features of a good pumping system are that it is capable of outputs of at least 50 MPa and ideally there must be no pulses (i.e. cyclical variations in pressure) as this may affect the detector response. There must be a flow capability of at least 10 cm3 min1 and up to 100 cm3 min–1 for preparative separations. Constant displacement pumps maintain a constant flow rate through the column irrespective of changing conditions within the column. The reciprocating pump is the most commonly used form of constant displacement pump. Such pumps produce small pulses of flow and pulse dampeners are usually incorporated into the system to minimise this pulsing effect. All constant displacement pumps have inbuilt safety cut-out mechanisms so that if the pressure within the column changes from pre-set limits the pump is inactivated automatically.

Detectors

Since the quantity of material applied to an HPLC column is normally very small, it is imperative that the sensitivity of the detector system is sufficiently high and stable to respond to the low concentrations of each analyte in the eluate. The most commonly used detectors are:

• Variable wavelength detectors: These are based upon ultraviolet–visible spectrophotometry. These types of detector are capable of measuring absorbances down to 190nm and can give full-scale deflection (AUFS) for as little as 0.001 absorbance units. They have a detection sensitivity of the order of 5 10–10 g cm–3 and a linear range of 105. All spectrophotometric detectors use continuous flow cells with a small internal volume (typically 8mm3) and optical path length of 10mm which allow the continuous monitoring of the column eluate.

• Scanning wavelength detectors: These have the facility to record the complete absorption spectrum of each analyte, thus aiding identification. Such opportunities are possible either by temporarily stopping the eluent flow or by the use of diode array techniques, which allow a scan of the complete spectrum of the eluate within 0.01 s and its display as a 3D plot on a VDU screen in real time.

• Fluorescence detectors: These are extremely valuable for HPLC because of their greater sensitivity (10–12 g cm–3) than UV detectors but they have a slightly reduced linear range (104). However, the technique is limited by the fact that relatively few analytes fluoresce. Pre-derivatisation of the test sample can broaden the applications of the technique.

• Electrochemical detectors: These are selective for electroactive analytes and are potentially highly sensitive. Two types are available, amperometric and coulometric, the principles of which are similar. A flow cell is fitted with two electrodes, a stable counter electrode and a working electrode. A constant potential is applied to the working electrode at such a value that, as an analyte flows through the flow cell, molecules of the analyte at the electrode surface undergo either an oxidation or a reduction, resulting in a current flow between the two electrodes. The size of the current is recorded to give the chromatogram. The potential applied to the counter electrode is sufficient to ensure that the current detected gives a full-scale deflection on the recorder within the working analyte range. The two types of detector differ in the extent of conversion of the analyte at the detector surface and on balance amperometric detectors are preferred since they have a higher sensitivity (10–12 g cm–3 as opposed to 10–8 g cm–3) and greater linear range (105 as opposed to 104).

GAS CHROMATOGRAPHY

The major components of a GC system are:

• a column housed in an oven that can be temperature programmed;

• a sample inlet point;

• a carrier gas supply and control; and

• a detector, amplifier and data recorder system

Columns

These are of two types:

• Packed conventional columns: These consist of a coiled glass or stainless steel column 13m long and 24mm internal diameter. They are packed with stationary phase coated on an inert silica support. Commonly used stationary phases include the polyethylene glycols (Carbowax 20M, very polar), methylphenyl- and methylvinylsilicone gums (OV17 and OV101, medium and non-polar respectively),Apiezon L (non-polar) and esters of adipic, succinic and phthalic acids. b-Cyclodextrinbased phases are available for chiral separations. The most commonly used support is Celite (diatomaceous silica), which because of the problem of support–sample interaction is often treated so that the hydroxyl groups that occur in the Celite are modified. This is normally achieved by silanisation of the support with such compounds as hexamethyldisilazane. The support particles have a large surface area and an even size, which, for the majority of practical applications, ranges from 6080 mesh (0.25mm) to 100120 mesh (0.125mm). The smaller the particle size and the thinner the coating the less band spreading occurs.

• Capillary (open tubular) columns: These are made of high-quality fused quartz and are 10100m long and 0.11.0mm internal diameter. They are of two types known as wall-coated open tubular (WCOT) and support-coated open tubular (SCOT), also known as porous layer open tubular (PLOT) columns, for adsorption work. In WCOT columns the stationary phase is thinly coated (0.15 mm) directly onto the walls of the capillary whilst in SCOT columns the support matrix is bonded to the walls of the capillary column and the stationary phase coated onto the support. Commonly used stationary phases include polyethylene glycol (CP wax and DB wax, very polar) and methyl and phenyl-polysiloxanes (BP1, non-polar; BP10, medium polar). They are coated onto the supporting matrix to give a 1% to 25% loading, depending upon the analysis. The capacity of SCOT columns is considerably higher than that of WCOT columns. The operating temperature for all types of column must be compatible with the stationary phase chosen for use. Too high a temperature results in excessive column bleed owing to the phase being volatilised off, contaminating the detector and giving an unstable recorder baseline. The working temperature range is chosen to give a balance between peak retention time and resolution. Column temperature is controlled to 0.1 oC. Analyte partition coefficients are particularly sensitive to temperature so that analysis times may be regulated by adjustment of the column oven, which can be operated in one of two modes:

• Isothermal analysis: Here a constant temperature is employed.

• Temperature programming: The temperature is gradually increased to facilitate the separation of compounds of widely differing polarity or Mr. This, however, sometimes results in excessive bleed of the stationary phase as the temperature is raised, giving rise to baseline variation. Consequently some instruments have two identical columns and detectors, one set of which is used as a reference. The currents from the two detectors are opposed, hence, assuming equal bleed from both columns, the resulting current gives a steady baseline as the column temperature is raised. The choice of phase for analysis depends on the analytes under investigation and is best chosen after reference to the literature.

Mobile phase

The mobile phase consists of an inert gas such as nitrogen for packed columns or helium or argon for capillary columns. The gas from a cylinder is pre-purified by passing through a variety of molecular sieves to remove oxygen, hydrocarbons and water vapour. It is then passed through the chromatography column at a flow rate of 4080 cm3 min–1. A gas-flow controller is used to ensure a constant flow irrespective of the back-pressure and temperature of the column.

Detectors

Flame ionisation detector (FID): This responds to almost all organic compounds. It has a minimum detection quantity of the order of 5 10–12g s1, a linear range of 107 and an upper temperature limit of 400 C. A mixture of hydrogen and air is introduced into the detector to give a flame, the jet of which forms one electrode, whilst the other electrode is a brass or platinum wire mounted near the tip of the flame. When the sample analytes emerge from the column they are ionised in the flame, resulting in an increased signal being passed to the recorder. The carrier gas passing through the column and the detector gives a small background signal, which can be offset electronically to give a stable baseline.

Mass spectrometer detector: This is a universal detector that gives a mass spectrum of the analyte and therefore gives both structural and quantitative data. Its detection limit is less than 1 ng per scan. Analytes may be detected by a total ion current (TIC) trace that is non-selective, or by selected ion monitoring (SIM) that can be specific for a selected analyte. In cases where authentic samples of the test compounds are not available for calibration purposes or in cases where the identity of the analytes is not known, a mass spectrometer is the best means of detecting and identifying the analyte. Special separators are available for removing the bulk of the carrier gas from the sample emerging from the column prior to its introduction in the mass spectrometer. Modern GC systems are controlled by dedicated microcomputers capable of automating and optimising the experimental conditions, recording the calibration and test retention data and carrying out statistical analysis of it and displaying the outputs in colour graphics in real time. They are capable of carrying out both qualitative and quantitative analysis on a similar basis to that of LC.