High Pressure Liquid Chromatography

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There are many ways to classify liquid column chromatography. ... In adsorption chromatography the stationary phase is an adsorbent (like silica gel or any.
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High Pressure Liquid Chromatography Y.C. Tripathi Rain Forest Research Institute, P. Box 136, Deovan, Sotai-Ali, A.T. Road, Jorhat–785 001 E-mail: [email protected]

In today’s science there is an ever growing need to visualize and characterize an ever increasing number of substances as we investigate our environment and isolate purify or create molecules and substances by numerous methods. High pressure liquid chromotography (HPLC) technology has evolved to offer numerous methods and analytical capabilities. HPLC as compared with the classical technique is characterized by: • Small diameter (2-5 mm), reusable stainless steel columns • Column packings with very small (3, 5 and 10 mm) particles and the continual development of new substances to be used as stationary phases • Relatively high inlet pressures and controlled flow of the mobile phase • Precise sample introduction without the need for large samples • Special continuous flow detectors capable of handling small flow rates and detecting very small amounts • Automated standardized instruments • Rapid analysis • High resolution. Initially, pressure was selected as the principal criterion of modern liquid chromatography and thus the name was “high pressure liquid chromatography” or HPLC. This was, however, an unfortunate term because it seems to indicate that the improved performance is primarily due to the high pressure. This is, however, not true. In fact high performance is the result of many factors: very small particles of narrow distribution range and uniform pore size and distribution, high pressure column slurry packing techniques, accurate low volume sample injectors, sensitive low volume detectors and of course, good pumping systems. Naturally, pressure is needed to permit a given flow rate of the mobile phase; otherwise, pressure is a

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negative factor not contributing to the improvement in separation. Recognizing this, most experienced chromatographers today refer to the technique as high performance liquid chromatography still permitting the use of the acronym HPLC.

TYPES OF HPLC There are many ways to classify liquid column chromatography. If this classification is based on the nature of the stationary phase and the separation process, three modes can be specified. In adsorption chromatography the stationary phase is an adsorbent (like silica gel or any other silica based packings) and the separation is based on repeated adsorption-desorption steps. In ion-exchange chromatography the stationary bed has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusively with ionic or ionizable samples. The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus the longer time it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time. In size exclusion chromatography the column is filled with material having precisely controlled pore sizes, and the sample is simply screened or filtered according to its solvated molecular size. Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the porous of the packing particles and elute later. Mainly for historical reasons, this technique is also called gel filtration or gel permeation chromatography although, today, the stationary phase is not restricted to a “gel”. Concerning the first type, two modes are defined depending on the relative polarity of the two phases: normal and reversed-phase chromatography. • In normal phase chromatography, the stationary bed is strongly polar in nature (e.g., silica gel), and the mobile phase is non-polar (such as n-hexane or tetrahydrofuran). Polar samples are thus retained on the polar surface of the column packing longer than less polar materials. • Reversed-phase chromatography is the inverse of this. The stationary bed is non-polar (hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. Here the more non-polar the material is, the longer it will be retained. Above mentioned types cover almost 90% of all chromatographic applications. Eluent polarity plays the highest role in all types of HPLC. There are two elution types: isocratic and gradient. In the first type constant eluent composition is pumped through the column during the whole analysis. In the second type, eluent composition (and strength) is steadily changed during the run.

Mobile Phases In HPLC, type and composition of the mobile phase (eluent) are one of the variables influencing the separation. Despite of the large variety of solvents used in HPLC, there are several common properties: • Purity • Detector compatibility

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• Solubility of the sample • Low viscosity • Chemical inertness • Reasonable price. Each mode of HPLC has its own requirements. For normal phase mode solvents are mainly nonpolar, for reversed-phase eluents are usually a mixture of water with some polar organic solvents such as acetonitrile. Size-exclusion HPLC has a special requirement, SEC eluents has to dissolve polymers, but the most important is that SEC eluent has to suppress all possible interactions of the sample molecule with the surface of the packing material.

STATIONARY PHASES (ADSORBENTS) HPLC separations are based on the surface interactions and depend on the types of the adsorption sites (surface chemistry). Modern HPLC adsorbents are the small rigid porous particles with high surface area. The main adsorbent parameters are: • Particle size: 3 to 10 µm • Particle size distribution: as narrow as possible, usually within 10% of the mean; • Pore size: 70 to 300 Å • Surface area: 50 to 250 m2/g • Bonding phase density (number of adsorption sites per surface unit): 1 to 5 per 1 nm2. The last parameter in represents an adsorbent surface chemistry. Depending on the type of the ligand attached to the surface, the adsorbent could be normal phase (–OH, –NH2), or reversed-phase (C8, C18, Phenyl), and even anion (NH4+), or cation (–COO–) exchangers.

DETECTORS Detectors equipped with the flow-through cell were a major breakthrough in the development of modern liquid chromatography. Such detection was first applied by the group of Tiselius, in Sweden in 1940, by continuously measuring the refractive index of the column effluent. Current LC detectors have wide dynamic range normally allowing both analytical and preparative scale runs on the same instrument. They have high sensitivities often allowing the detection of nanograms of material, and the better models are very flexible, allowing rapid conversion from one mobile phase to another and from one mode to another. Almost all LC detectors are the on-stream monitors. The only relatively successful off-line detector is FTIR spiral disk monitor, which require sample transfer on the germanium disk and scanning in FTIR instrument. HPLC detectors always used under continuous flow conditions and the sample is always dissolved in the eluent during detection. Actual sample is only present in a ng quantity in the detector, but in trace analysis, this quantity could be fg and even the single molecule. The mobile phase is a factor which must always be considered. In the last decade there is a significant progress in the development of LC/MS interfacing systems. MS as an on-line HPLC detector is said to be the most sensitive, selective and in the same time the most universal detector. But it is still the most expensive one. Following are some of the important detectors used in HPLC systems:

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Most common Refractive index • UV/VIS • Fixed wavelength • Variable wavelength • Diode array • Fluorescence

Less common, but important • Conductivity • Mass-spectrometric (LC/MS) • Evaporative light scattering

BASIC DETECTOR REQUIREMENTS Regardless of the principle of operation, an ideal LC detector should have the following properties: • Low drift and noise level (particularly crucial in trace analysis). • High sensitivity. • Fast response. • Wide linear dynamic range (this simplifies quantitation). • Low dead volume (minimal peak broadening). • Cell design which eliminates remixing of the separated bands. • Insensitivity to changes in type of solvent, flow rate, and temperature. • Operational simplicity and reliability. • It should be tuneable so that detection can be optimized for different compounds. • It should be non-destructive.

Noise and Drift HPLC is a time-dependent process. The appearance of the component from the column in the detector is represented by the deflection of the recorder pen from the baseline. It is a problem to distinguish between the actual component and artifact caused by the pressure fluctuation, bubble, compositional fluctuation, etc. If the peaks are fairly large, one has no problem in distinguishing them. However, the smaller the peak, the more important is that the baseline be smooth, free of noise, and drift. Fig. 1 illustrates noise and drift levels of detector. Baseline noise is the short time variation of the baseline from a straight line caused by electric signal fluctuations, lamp instability, temperature fluctuations and other factors. Noise usually has much higher frequency than actual chromatographic peak. Noise is normally measured “peak-to-peak” i.e., the distance from the top of one such small peak to the bottom of the next. Sometimes, noise is averaged over a specified period of time. Noise is the factor which limits detector sensitivity. In trace analysis, the operator must be able to distinguish between noise spikes and component peaks. A practical limit for this is a 3 x signal-to-noise ratio, but only for qualitative purposes. Practical quantitative detection limit better be chosen as 10x signal-to-noise ratio. This ensures correct quantification of the trace amounts with less than 2% variance. Another parameter related to the detector signal fluctuation is drift. Noise is a short-time characteristic of a detector, an additional requirement is that the baseline should deviate as little as possible

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Fig. 1. Definition of noise, drift and smallest detectable peak.

from a horizontal line. It is usually measured for a specified time, e.g., 1/2 hour or one hour. Drift usually associated to the detector heat-up in the first hour after power-on.

SENSITIVITY Detector sensitivity is one of the most important properties of a LC detector. Sensitivity of the detector is a measure of its ability to discriminate between small differences in analyte concentration. So, it is actually the slope of the calibration curve. It is also dependent on the standard deviation of the measurements. The higher the slope of your calibration curve the higher the sensitivity of your detector for that particular component, but high fluctuations of your measurements will decrease the sensitivity. Sensitivity of a detector is not the minimum amount that can be detected. This value is influenced by the chromatographic conditions. Early eluting peaks are usually sharp, whereas the ones with long retention times are broad and sometimes difficult to discern from the noise.

SELECTIVITY Selectivity is another highly desirable property of HPLC detectors. A selective detector allows one to see only components of interest despite of their co-elution with any others. Refractive index is an example of almost nonselective detector. Any component could make a response, but in case of poorly resolved mixture analyst will not be able to distinguish components. Fluorescence and electrochemical detectors are the most selective among the common detectors. Only about 10% of organic compounds are able to fluorescence, and by choosing excitation and emission wavelength specific for the particular compound one can detect only this compound. Usually, the more selective the detection, the lower is the signal noise and the higher the sensitivity.

RESPONSE The definition of detector response depends on whether it is mass-sensitive or concentrationsensitive. For mass-sensitive detectors, the response R (mV/mass/unit time) is given by the following relationship: R=

hw sM

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For the concentration sensitive detector, sensitivity is given in units of mV/mass/unit volume and it can be calculated using the following formula:

hwF R = sM where, h = peak height (mV) w = peak width at 0.607 of the peak height (cm) F = flow rate (ml/min) M = mass of solute injected s = chart speed (cm/min) In addition to factors such as the nature of the mobile phase, cell geometry, and so on, the detector response is also a function of the type of solute.

Linear Dynamic Range The detector response is said to be linear if the difference in response for two concentrations of a given compound is proportional to the difference in concentration of the two samples. Such response appears as a straight line in the calibration curve (Fig-2). The linear dynamic range of a detector is the maximum linear response divided by the detector noise. Most detectors eventually become non-linear as sample size is increased and this upper point is usually well defined. The chromatographer should know where this occurs to avoid errors in quantification.

Fig. 2.

Detector response.

Cell Efficiency The heart of an efficient HPLC detector is the cell. Fig. 3 shows the schematic of a modern follow-through cell. The optics provides focusing of the light beam in the center of the cell where it is virtually unaffected by the entry-exit-window-interface disturbance, or drift induced by flow, temperature, or refractive index changes. The short, wide cell assures that maximum energy is transmitted, and a post-cell collecting lens focuses all of the existing light from the cell onto the photo-detector. Modern LC systems provide high resolution in a short

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Fig. 3. Schematic of a modern follow-through cell.

time. For example, if we are using a column which provides 10,000 theoretical plates and has 15 cm length, then for the component eluted in 2 min (2 ml at 1 ml/min flow rate) we will ideally have peak width of 80 µl. Thus, having the flow-cell volume of 20 µl we will have only 4 independent measurements on this peak. This is definitely not enough to correctly describe peak shape and will introduce apparent peak broadening. Another important feature of the flow-cell is to compensate on the refraction effect. When the components of your mixture pass though the cell they actually change (slightly) the eluent composition which will change the refraction coefficient. If the light beam is not parallel the change in the refraction will change light scattering and will contribute into the apparent adsorption readings. Unfortunately very few cell designs have this compensation. • Apparent Peak broadening happens if the flow cell volume is more then 1/10 of the peak volume, or if the cell geometry is not optimized. • Since the peak area is proportional to the amount of the injected analyte, the peak broadening will cause the reduction of the peak height. In case of apparent peak broadening due to the big cell volume but if the cell geometry is optimized we may not see a significant height reduction. • The resolution may be decreased as a result of the peak broadening and peak overlapping occurs. • If the cell geometry is not optimized one can notice an appearance of peak tailing. This indicates the presence of flow stagnant zones.

REFRACTIVE INDEX DETECTOR The refractive index (RI) detector is the only universal detector in HPLC. The detection principle involves measuring of the change in refractive index of the column effluent passing through the flow-cell.  The greater the RI difference between sample and mobile phase, the

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larger the imbalance will become. Thus, the sensitivity will be higher for the higher difference in RI between sample and mobile phase. On the other hand, in complex mixtures, sample components may cover a wide range of refractive index values and some may closely match that of the mobile phase, becoming invisible to the detector. RI detector is a pure differential instrument, and any changes in the eluent composition require the rebalancing of the detector. This factor is severely limiting RI detector application in the analyses requiring the gradient elution, where mobile phase composition is changed during the analysis to effect the separation. There are two basic types of RI detectors that require use of a two-path cell where the sample-containing side is constantly compared with the non-sample-containing reference side.

Deflection Detectors This detector based on the deflection principle of refractometery, where the deflection of a light beam is changed when the composition in the sample flow-cell changes in relation to the reference side (as eluting sample moves through the system). When no sample is present in the cell, the light passing through both sides is focused on the photodetector (usually photoresistor). The optical schematic of the deflection detector is shown in Fig. 4. Sample cell Incident beam

eam

Deflected b

Reference cell

Fig. 4.

Optical schematic of a deflection RI detector.

As sample elutes through one side, the changing angle of refraction moves the beam. This results in a change in the photon current falling on the detector, which unbalances it. The extent of unbalance (which can be related to the sample concentration) is recorded on a strip chart recorder. The advantages of this type of detector are: (1) universal response; (2) low sensitivity to dirt and air bubbles in the cells; and (3) the ability to cover the entire refractive index range from 1.000 to 1.750 RI with a single, easily balanced cell. The disadvantages are the relatively low sensitivity and a general disability to easily remove and clean or replace the cell when filming or clogging occurs.

Reflective Detectors The refractive index detector based on the Fresnel principle is relatively rear. There was only one or two commercial models and they are not in the production any more. Fig. 5 shows the optical schematic of this type of detector. Here, the light beam is reflected from the liquid-

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Fig. 5. Fresnel-type RI detector.

glass interface in the detecting photocell. The introduction of sample into one cell causes light to be refracted at a different angle. The deflection of the light beam from the photoresistor causes the appearance of the electrical signal. Here, too, this difference between sample cell signal and reference-cell signal is output to a recorder or data handling system as peak. The major advantage of this type of detector is a very high sensitivity since the optics allows a higher concentration of signal in a particular RI range than is possible in other widerange detectors. Other advantages include the ability to operate at extremely low flow rates with very low-volume cells, easy cell accessibility, and low cost. Its disadvantages are the incredible sensitivity to the flow and pressure fluctuations and the need for changing prisms to accommodate either high or low RI solvents and to manually adjust the optical path when making solvent changes. The refractive index of an analyte is a function of its concentration. Change in concentration is reflected as a change in the RI.  A refractive index detector for liquid chromatography should be sensitive to changes as small as 10–7 RI units (corresponding to a concentration change of 1 ppm). Presence of dissolved air, changes in solvent composition, improper mixing and column bleed will contribute to baseline drift. Eluent pressure change of 15 psi will cause the change of 1 x 10–6 RI unit and 1°C temperature variation will be equivalent to the change of 600 x 10–6 RI units. Thus it is obvious that both of these parameters must be closely controlled, especially temperature. To operate at high sensitivities, a RI detector must usually be thermostated (± 0.01°C), actually the using of the water bath connected to the detector head does not give required temperature stability and alternately passive thermostabilization with massive metallic block usually gives much better results.

ULTRAVIOLET/VISIBLE SPECTROSCOPIC DETECTORS Any chemical compound could interact with the electromagnetic field. Beam of the electromagnetic radiation passed through the detector flow-cell will experience some change in its intensity due to this interaction. Measurement of this change is the basis of the most optical HPLC detectors. Radiation absorbance depends on the radiation wavelength and the functional groups of the chemical compound. Electromagnetic field depending on its energy (frequency) can interact with electrons causing their excitation and transfer onto the higher

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energetical level, or it can excite molecular bonds causing their vibration or rotation of the functional group. The intensity of the beam which energy corresponds to the possible transitions will decrease while it is passing through the flow-cell. According to the LambertBear law absorbance of the radiation is proportional to the compound concentration in the cell and the length of the cell. The electromagnetic spectrum is traditionally divided mainly into four regions viz., Infrared (IR) (2,500 – 50,000 nm); Near IR (800 – 2,500 nm); Visible (400 – 800 nm) and Ultraviolet (190 – 400 nm). Three major regions (IR, visible, and UV) are used in the spectroscopy. In liquid chromatography, IR spectrophotometers have found only limited use. There are few transparent polar liquids which can be used as the mobile phase. On the other hand, spectrophotometers working in the range (200 – 600 nm) are used widely as LC detectors. UV and visible region of the electromagnetic radiation corresponds to the excitation of the relatively low energy electrons such as pi-electrons, or non-paired electrons of some functional groups. For example, n-alkanes could absorb in the UV region below 180 nm. I-electrons require high energy radiation to get excited and to show absorption of the radiation. But any compounds which have benzene ring will show absorbance at 205–225 and 245–265 nm. The last corresponds to the excitation of conjugated p-electrons of the benzene ring. The majority of organic compounds can be analyzed by UV/VIS detectors. Almost 70% of published HPLC analyses were performed with UV/VIS detectors. This fact, plus the relative ease of its operation, makes the UV detector the most useful and the most widely used LC detector.

UV ABSORBANCE Absorbance is the logarithm of the ratio of the intensities of the incident light (Io) and the transmitted light (I). It is related according to the Beer-Lambert Law to the molar absorptivity (molar extinction coefficient, A), the thickness of the substance (i.e., the path length of the cell, b) and the molar concentration of the substance (c): A = log

FG I IJ = ebc HIK o

In HPLC, the photodetector measures transmitted light I, but the electronics converts this signal to a logarithmic relationship (A) which is proportional to concentration. The ordinate of the chromatogram represents the detector signal, which in general, is proportional to the analyte concentration in the cell. Since chromatographic systems permit the quantitative analysis of sample components representing many orders of magnitude—from ppm to percent concentrations, one may select various amplification ranges for visual display of components (both small and large). In UV detection, one expresses the detector range in absorbance units (A). One absorbance unit corresponds to the depreciation of the light intensity by 90% of the incident light. Molar Absorptivity: This term is also called the molar extinction coefficient—corresponds to the absorbance for a molar concentration of the substance with a path length of 1 cm. Molar adsorptivity is dependent on the wavelength and chromatographic conditions (solvent, pH and temperature). It is a constant at a specified wavelength. Table 1 lists the molar absorptivities of a number of compounds at specified wavelengths.

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Table 1. Molar Absorptivity (e) values of various compound types at specified wavelengths Name Acetylide Aldehyde Amine Azo Bromide Carboxyl Disulphide Ester Ether Ketone Nitrate Nitrile Nitrite Nitro

Chromophore

Wavelength [nm]

–C=C –CHO –NH2 –N=N–Br –COOH –S-S–COOR –O>C=O –ONO2 –C=N –ONO –NO2

175-180 210 195 285-400 208 200-210 194 205 185 195 270 160 220 - 230 210

Molar extinction, e 6,000 1,500 2,800 3-25 300 50 - 70 5,500 50 1,000 1,000 12 1000-2000 strong

FIXED WAVELENGTH DETECTORS HPLC detectors which do not allow changing the wavelength of the radiation called fixedwavelength detectors. Low-pressure mercury vapor lamps emit very intense light at 253.7 nm. By filtering out all other emitted wavelengths, manufacturers have been able to utilize this 254 nm line to provide stable, highly sensitive detectors capable of measuring subnanogram quantities of any components which contains aromatic ring. The 254 nm was chosen since the most intense line of mercury lamp is 254 nm, and most of UV absorbing compounds have some absorbance at 254 nm.

VARIABLE WAVELENGTH DETECTORS Detectors which allow the selection of the operating wavelength called variable wavelength detectors (Fig. 6) and they are particularly useful in three cases: • Offer best sensitivity for any absorptive component by selecting an appropriate wavelength; • Individual sample components have high absorptivity at different wavelengths and thus, operation at a single wavelength would reduce the system’s sensitivity; • Depending on the sophistication of the detector, wavelength change is done manually or programmed on a time basis into the memory of the system.

DIODE-ARRAY DETECTORS Diode array adds a new dimension of analytical capability to liquid chromatography because it permits qualitative information to be obtained beyond simple identification by retention time (Fig. 7).

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Memory lamp Photodiode

Amplifier

Flow cell

Fig. 6A.

Fixed wavelength detector. Reference photodiode

Beam splitter

Duterium lamp Mirror

Slit Comparator Grating

Sample cell

Photodiode Amplifier

Fig. 6B.

Variable-wavelength detectors.

Fig. 7. Diode-array detectors.

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There are two major advantages of diode array detection. In the first, it allows for the best wavelength(s) to be selected for actual analysis, which is particularly important when molar absorptivities at different wavelengths are not known. The second major advantage is related to the problem of peak purity. Often, the peak shape in itself does not reveal that it actually corresponds to two (or even more) components. In such a case, absorbance rationing at several wavelengths is particularly helpful in deciding whether the peak represents a single compound or, is in fact, a composite peak. In absorbance rationing, the absorbance is measured at two or more wavelengths and ratios are calculated for two selected wavelengths. Simultaneous measurement at several wavelengths allows one to calculate the absorbance ratio. The ratios at chosen wavelength are continuously monitored during the analysis; if the compound under the peak is pure, the response will be a square wave function (rectangle). If the response is not rectangle, the peak is not pure.

FLUORESCENCE DETECTORS Fluorescence detectors are probably the most sensitive among the existing modern HPLC detectors. It is possible to detect even a presence of a single analyte molecule in the flow cell.  Typically, fluorescence sensitivity is 10–1000 times higher than that of the UV detector for strong UV absorbing materials. Fluorescence detectors (Fig. 8) are very specific and selective among the others optical detectors normally used as an advantage in the measurement of specific fluorescent species in samples. When compounds having specific functional groups are excited by shorter wavelength energy and emit higher wavelength radiation which called fluorescence. Usually, the emission is measured at right angles to the excitation. Roughly about 15% of all compounds have a natural fluorescence. The presence of conjugated pi-electrons especially in the aromatic components gives the most intense fluorescent activity. Also, aliphatic and alicyclic compounds with carbonyl groups and compounds with highly conjugated double bonds fluoresce, but usually to a lesser degree.

Fig. 8. A typical Fluorescence Detector.

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Most unsubstituted aromatic hydrocarbons fluoresce with quantum yield increasing with the number of rings, their degree of condensation and their structural rigidity. The detectors differ in the method in which the wavelengths are controlled. Less expensive instruments utilize filters; medium priced units offer monochromator control of at least emission wavelength, and full capability research-grade instruments provide monochromator control of both excitation and emission wavelengths. Fluorescence intensity depends on both the excitation and emission wavelength, allowing selectively detect some components while suppressing the emission of others. The detection of any component significantly depends on the chosen wavelength and if one component could be detected at 280 ex and 340 em, another could be missed. Most of the modern detectors allow fast switch of the excitation and emission wavelength, which offer the possibility to detect all component in the mixture. For example, in the very important polynuclear aromatic chromatogram the excitation and emission wavelengths were 280 and 340 nm, respectively, for the first 6 components, and then changed to the respective values of 305 and 430 nm; the latter values represent the best compromise to allow sensitive detection of compounds.

ELECTROCHEMICAL DETECTORS The electrochemical detector (Fig. 9) is also a popular liquid chromatographic detector having additional selectivity and sensitivity for some compounds. It is based on the measurements of the current resulting from oxidation/reduction reaction of the analyte at a suitable electrode. Since the level of the current is directly proportional to the analyte concentration, this detector could be used for quantification. The eluent should contain electrolyte and be electrically conductive. The purity of the eluent is very important, because the presence of oxygen, metal contamination and halides may cause significant background current and therefore, noise and drift in the base line. Most of the analytes to be successfully detected require the pH adjustments.

Fig. 9.

Electrochemical Detectors.

The areas of application of electrochemical detection are not large, but the compounds for which it does apply, represent some of the most important drug, pollutant and natural product classes. For these, the specificity, and sensitivity make it very useful for monitoring these compounds in complex matrices such as body fluids and natural products. Sensitivities for compounds such as phenol, catecholamines, nitrosamines, and organic acids are in the picomole (nanogram) range.

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ELECTROLYTIC CONDUCTIVITY DETECTOR The conductivity of the column effluent is continuously measured and the appearance of the analyte in the cell is indicated by a change in conductivity. Usually this is a very low volume flow-through capillary equipped with two electrodes and variations in conductivity of the mobile phase due to the eluted sample components are continuously recorded (Fig. 10). Response is linear with concentration over a wide range; quantitation of the output signal is possible with suitable preliminary calibration. Best use is made of this detector in isocratic analysis since solvent gradients will cause a proportional shift in the baseline.

Fig. 10. Electrochemical Detectors.

Such detectors have been used most successfully in ion-exchange chromatography of anions and cation but generally, they have found only limited popular acceptance.

EVAPORATIVE LIGHT SCATTERING DETECTORS (ELSD) Commonly described as a poor man’s Mass Spectrometer, this unique type of detector offers amazing capabilities to measure a wide variety of substances. Many substance types do not produce any UV-VIS absorbance’s and cannot be observed on typical UV based HPLC detection systems. The ELSD detector is basically quite similar to an electrospray part of an electrospray ESI Mass Spectrometer. In that there is a heated chamber and a flow of a carrier dispersing gas such as high grade Argon or ultra pure Nitrogen gas that served to atomize the HPLC eluent liquid mobile phase flow upon arrival and the atomized spray is then passed through a detector with a laser energy pulse that excites the molecules which in turn scatters the light. The light scattering is measured by a high sensitivity or series of detectors and compared to a reference beam measurement. Unlike ESI Mass Spectrometers however, there is absence of a high vacuum system and time of flight tube that facilitate mass measurement. There are companies that have developed instruments that can convert ELSD data to calculate molecular mass. Yet the mass accuracy is reported as good for rough estimations of molecular weights, there are many variables and the mass accuracy is significantly lower than that achievable via true mass spectrometry. For example such a system may assign a mass of 56Kd to a protein but the actual mass of the protein may be 52 Kd so it certainly good for screening and basic evaluations of HPLC peak isolates.

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The advantages of ELSD are that it allows visualization of certain types of molecules that for example do not have UV Absorbance’s these include types of Glycosides. Further advantage is that it can provide ballpark mass determinations of peak isolates by comparison to results obtained running known molecular weight standards producing more accurate results than Electrophoresis running molecular weight standards.

CHEMICAL ION DETECTORS These detectors utilize ion selective electrodes that are implanted into the flow cell to measure specific ions from substances that elute from the HPLC column. This measurement is greatly influenced by presence of various metal ions and requires either Teflon HPLC pump heads and use of “Peak” plastic tubing’s and or special in-line metal trapping filters as the metal ions will interfere with measurement of target ions via quenching of signals or via poor signal to noise ratios and suboptimal detection.

HPLC-MASS SPECTROMETRY (LC-MS) The most common type of LC-MS is via conversion of an electro-spray system to allow sample introduction via micro-flow HPLC to the electro-spray atomization chamber versus direct sample introduction via a syringe pump system. LC-MS allows that an optimized HPLC separation be effected to separate different components allowing their discrete analysis by mass ionization. LC-MS systems typically utilize either a photodiode array UV-Scanning detector or a UV detector between the column outflow and the atomizer chamber. Thus, both the absorbance trace and the mass spectra data are observed and recorded. Through various softwares most often from system manufacturers, the HPLC chromatogram peak isolates shown from the HPLC detector can be mass labeled as well as labeled with the elution time and the text report provides peak area. In the case of systems with a photodiode array scanning UV-VIS detector the absorbance’s spectra for all significant wavelength absorbance’s is plotted either via color plotting using different colors or via solid, dashed or dotted etc. lines in the case of monochrome. Thus, the absorbance maxima for unknown UV-VIS light absorbing compounds, the elution time, the peak area’s at various wavelengths and the mass of the eluted peak are all determined and present in the data. Examination of both peak symmetry and various absorbances determine the relative purity of eluted compounds. Peak area compared to that of standards allows analytical quantitation and mass analysis determines the molecular weight of each eluted compound isolate.

HPLC SEPARATION TECHNOLOGY The separation of components, molecules, substances and analytes is the goal of a chromatographic separation process. This goal is achieved by careful analytical work in the choice and preparation of the mobile phase liquid solvent buffers, choice of the type of solid phase column, and choice of the gradient conditions between buffer solvents. The choice of flow rate, depth of gradient, slope of gradient, column particle size and hydrophobic interaction solid phase type are combined with sample measurement and introduction choices, column heater temperature control and the type of detector and its settings and type of data acquisition utilized to produce analytical results.

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METHOD DEVELOPMENT While performing HPLC on any type or class of materials, the first aspect is thorough examination of scientific literature about these materials and specifically evaluating published accounts of their separations characteristics. In many cases various methods advised to be used as comparing the actual chromatograms may provide great insight for choosing the best method. For unknown or novel compounds it may require that numerous runs be made with different mobile phase solvents, different gradient types, different column types, and even different detector types. The literature search for the type of most similar compounds results will act as a guide to narrow down the number of different methods to run and compare.

METHOD OPTIMIZATION The separation can be optimized by a complete methods development with particular attention to adjusting the gradient slope and time making gradients more shallow or steep will effectively zoom-in on the region of buffer solvent mobile phase concentrations and allow the degree of gradient change (% organic solvent concentration) to change more slowly effecting better separation of individual constituents.

BASIS OF SEPARATION PROCESS Chromatographic separation process based on the difference in the surface interactions of the analyte and eluent molecules. Let us consider a separation of a two component mixture dissolved in the eluent. Assume that component A has the same interaction with the adsorbent surface as an eluent, and component B has strong excessive interaction. Being injected into the column, these components will be forced through by eluent flow. Molecules of the component A will interact with the adsorbent surface and retard on it by the same way as an eluent molecules. Thus, as an average result, component A will move through the column with the same speed as an eluent. Molecules of the component B being adsorbed on the surface (due to their strong excessive interactions) will sit on it much longer. Thus, it will move through the column slower than the eluent flow. A general shape of the chromatogram for this mixture is presented in Fig. 11. Usually a relatively narrow band is injected (5 - 20 µl injection volume). During the run, the original chromatographic band will be spread due to the noneven flows around and inside the porous particles, slow adsorption kinetics, longitudinal diffusion and other factors. These

Fig. 11.

A General chromatogram.

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processes together produce so called band broadening of the chromatographic zone. In general, the longer the component retained on the column, the broader its zone. Separation performance depends on both component retention and band broadening. Band broadening is, in general, a kinetic parameter, dependent on the adsorbent particle size, porosity, pore size, column size, shape, and packing performance. On the other hand, retention does not depend on the above mentioned parameters, but it reflects molecular surface interactions and depends on the total adsorbent surface.

RETENTION PARAMETERS The easiest way to find the chromatographic retention is to measure the time between the injection point and maximum of the detector response for correspondent compound. This parameter usually called “retention time”. Retention time, tR is inversely proportional to the eluent flow rate. The product of retention time and eluent flow rate, so called “retention volume”, is more of a global retention parameter. Retention volume, VR represents the volume of the eluent passed through the column while eluting a particular component. Component retention volume VR could be split into two parts: 1. Reduced retention volume is the volume of the eluent that passed through the column while the component was sitting on the surface. 2. Dead volume is the volume of the eluent that passed through the column while the component was moving with the liquid phase. The second part is equal to the volume of the liquid phase in the column (dead volume, Vo), and it will be the same for any component eluted on this column. Retention volume is independent of the flow parameters for the particular run, but it depend on the geometrical parameters of the column. VR will be different for the same compound eluted on the different columns packed with the same type of adsorbent. The more universal and fundamental retention parameter (k) is the ratio of the retention volume and dead volume (k = VR / VO). Historically, a slightly different retention parameter, called “capacity factor” (k¢) was introduced by the analogy with the liquid partitioning theory and widely accepted in chromatographic practice (k¢ = (VR – VO) / VO). Capacity factor is dimensionless and independent on any geometrical parameters of the column or HPLC system. It could be considered to be a thermodynamic characteristic of the adsorbent-compound-eluent system.

BAND BROADENING (COLUMN EFFICIENCY) After injection, a narrow chromatographic band is broadened during its movement through the column. The higher the column band broadening, the smaller is the number of components that can be separated in a given time. In other words, the sharpness of the peak is an indication of how good or efficient a column is. The peak width is an indication of peak sharpness and, in general, an indication of the column efficiency. However, the peak width is dependent on a number of parameters (column length, flow rate, particle size). Flow rate is the only parameter which can be changed from run to run on the same column. Thus, it is better to consider a relative value to express column efficiency. In absence of the specific interactions or sample overloading, the chromatographic peak can be represented by a Gaussian curve with the standard deviation(s) (Fig. 12). The ratio

High Pressure Liquid Chromatography

Fig. 12.

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Gaussian curve.

of standard deviation to the peak retention time (s/tR) is called the relative standard deviation, which is independent on the flow rate. In practice, the square of the reciprocal value is normally used N – (tR/I)2. This has become the accepted expression of column efficiency. The reason for using the second power has come from statistics, and it is related to the fact that not the standard deviation (I), but its square, the variance (I)2 is the basic measure of normal distribution. The value N is called the plate number or the number of theoretical plates. The term “theoretical plate” came form the analogy with the distillation theory. In practice, it is more convenient to measure peak width either at the base line, or at the half eight, and not at 0.609 of the peak height, which actually correspond to 2I. N = 16 (tR/Wb)2 ; N = 5.545 (tR/Wb)2 The plate number depends on column length: the longer the column, the larger the plate number. Therefore, the plate height term has been introduced to measure how efficiently column has been packed, h = L/N. The lower the plate height and the higher the plate number, the more efficient will be the chromatographic column. It is well recognized now that column band broadening originates from three main sources viz., multiple path of an analyte through the column packing; molecular diffusion and effect of mass transfer between phases. The velocity of mobile phase in the column may vary significantly across the column diameter, depending on the particle shape, porosity, and the whole bed structure. Band broadening is caused by differing flow velocities through the column. Molecules disperse or mix due to the diffusion. The longitudinal diffusion (along the column long axis) leads to the band broadening of the chromatographic zone. The higher the eluent velocity, the lower the diffusion effect on the band broadening. Molecular diffusion in the liquid phase is about five orders of magnitude lower than that in the gas phase, thus this effect is almost negligible at the standard HPLC flow rates.

SELECTIVITY AND RESOLUTION Selectivity is the ratio of the capacity factors of both peaks, or the ratio of its adjusted retention times. Selectivity represents the separation power of particular adsorbent to the mixture of these particular components. = = VR,1 – VO / VR,2 – VO = K¢1 / K¢2

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This parameter is independent of the column efficiency; it only depends on the nature of the components, eluent type, eluent composition, and adsorbent surface chemistry. In general, if the selectivity of two components is equal to 1, then there is no way to separate them by improving the column efficiency. Resolution is the parameter describing the separation power of the complete chromatographic system relative to the particular components of the mixture. By convention, resolution (R) is expressed as the ratio of the distance between two peak maxima to the mean value of the peak width at the baseline: R = 2 x VR,2 – V R,1 / W1 + W2 If peaks are approximated by symmetric triangles, then, if R is equal to or more than 1 then component are completely separated. If R is less than 1, then components are overlapped.

KINETICS OF MASS TRANSFER Mass transfer is the most questionable parameter. For the modern types of packing materials it may combine two effects: adsorption kinetics and mass transfer (mainly due to diffusion) inside the particles. 95% of all modern packing materials are the spherical, totally porous rigid particles with average diameter ~5 µm and pore diameter ~100Å. Ratio of the particle to the pore diameter is 500/1. There is no pressure propelled flow inside the particle, and molecules can move there only by diffusion. It can be shown by analogy: if consider a tunnel in the mountain which has diameter of 2 meters and one kilometer length (same ratio 500/1), and if there is a storm outside with 200 km/h wind, there will be almost no wind in 50 meters from tunnel entrance.

THERMODYNAMICS OF HPLC RETENTION There are two basic approaches for thermodynamic description of the HPLC retention phenomena, one is based on the partitioning theory and another is based on adsorption. Partition is a concentrational change in the system due to the distribution of the components between two (or more) phases. Adsorption is the concentrational changes in the system in presence of interface with another phase and due to the surface forces. Phase is a form of matter that is uniform throughout in chemical composition and physical state. In HPLC we have two main phases viz., the mobile phase (eluent) which is a liquid solvent or mixture of solvents which is moving through the chromatographic column and carrying analytes and the stationary phase (adsorbent), solid porous media consisting of the rigid porous particles, usually silica based, with the specific surface properties. Adsorbent particles are considered to be nonpermeable and nonsoluble for the eluent and analyte molecules. It only introduces surface forces in the system. Consideration of the HPLC process based on the partitioning theory was transferred from GC (gas chromatography) theory, where we usually have a mobile gas phase and stationary liquid phase, and where a true partitioning occurs. Usual description of liquid chromatography on the partitioning basis considers the assumption of the existence of the separate liquid phase which is close to the adsorbent surface. Chemically bonded phases are usually considered by this manner. The most popular bonded phase is octadecylsilica, where relatively long (21 Å) alkyl chains are chemically bonded to the

High Pressure Liquid Chromatography

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silica surface. The main partitioning concept is that analyte molecules can penetrate between these alkyl chains. This process thermodynamically considered as dissolving of the analyte molecules in the surface alkyl phase. Two restrictions exist for application of this approach in thermodynamic description i.e., the bonded chains have a certain thickness (about 3.8 Å in diameter) and depending on the bonding density the layer may not behave as a liquid and monomolecular layer would not be considered a phase in classical thermodynamics.

Adsorption from Solutions A classic description of the adsorption process is based on the Gibbs excess adsorption theory, which basically considers two similar hypothetical adsorption systems with the same volume, temperature, pressure, and adsorbent surface area. The only difference is that the first system does not show any adsorption on the surface and the second does.

EFFECT OF TEMPERATURE Temperature effects in HPLC are not as significant as in gas chromatography as temperature range is not always the same. Volatile solvents are not allowed to rise to higher temperatures too much, and the stability of the attached bonded ligands on the adsorbent surface may be influenced by the high temperature. So, the main temperature range is from ambient temperature to 60 or 70 C. Increasing the temperature will decrease the value of K or k’, thus the actual retention time will decrease. For most of the systems these decrease will not exceed 50% of the component reduced retention time at ambient temperature. There are two other significant effects of separation under the elevated temperature. (i) Stabilization of the column under the elevated temperature usually leads to the stabilization of the retention times. (ii) Another effect is the increase of the column efficiency. At the elevated temperature viscosity of liquids decrease and the diffusion coefficient increase.

MOBILE PHASE EFFECT In most of the HPLC separations binary eluents are employed. One of the solvents in the eluent is usually inert relative to the surface interactions. In Reversed-phase HPLC (RP HPLC) one of the eluent components is water, which does not interact with the hydrophobic adsorbent surface. And it does not compete with the analyte for the adsorption sutes. In normal-phase HPLC (NP HPLC) one of the eluent components is usually hexane, which also does not interact with the very polar silica surface. Another component of any binary eluent is an active one. It usually called a “modifier” because it can interact with the adsorbent surface and compete with analyte molecules for the adsorption sites. Increasing of the concentration of the “modifier” in the eluent leads to the decreasing of the analytes retention. Capacity factor is proportional to the thermodynamic equilibrium constant, and the last is an exponent of the free Gibbs energy of the system For the binary eluent system, only the “modifier” can interact with the surface. For RP HPLC it will be an organic component of the eluent, and water is assumed not to interact with the surface.

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EFFECT OF pH A general approach to the separation of the mixtures containing an ionisible components is to suppress their ionization. Suppression of the ionization decreases a power of the molecular solvation and exposes the hydrophobic (organic) part of the molecule to the surface interaction. Ionization suppression is usually made by the adding a buffer into the solvent, which shift a pH to the certain value. In the absence of buffer, easy ionizible components are eluted from the column as very broad peaks. According to the Le Chatelier principle, dissolved ionizible component is present in the solution as a mixture of ions and nonionized molecules [AB] = [A+] + [B–] According to the above equilibrium, about 50% of all molecules are ionized in the solution. But, the chromatographic behavior of ions and neutral molecules are different. Let us assume that neutral molecules will be retained, so during the run ions will move faster, and at the first moment they will be separated from the neutral molecules. But, according to the above equilibrium, in the absence of the neutral molecules ions will tend to form them, and this new neutral molecule will also be absorbed, and so on. This process will lead to the spreading of the component along the column and causes the appearance of the broad peak. It does not occur if the equilibrium is shifted due to the presence of the buffer with the pH at least two units apart of pK of the component.

RETENTION MECHANISM In general, HPLC is a dynamic adsorption process. Analyte molecules, while moving through the porous packing bead, tend to interact with the surface adsorption sites. Depending on the HPLC mode, the different types of the adsorption forces may be included in the retention process: • Hydrophobic interactions are the main ones in reversed-phase separations. • Dipole-dipole (polar) interactions are dominated in normal phase mode. • Ionic interactions are responsible for the retention in ion-exchange chromatography. All these interactions are competitive. Analyte molecules are competing with the eluent molecules for the adsorption sites. So, the stronger analyte molecules interact with the surface, and the weaker the eluent interaction, the longer analyte will be retained on the surface. Size-exclusion chromatography (SEC) is a special case. It is the separation of the mixture by the molecular size of its components. In this mode any positive surface interactions should be avoided (eluent molecules should have much stronger interaction with the surface than analyte molecules). Basic principle of SEC separation is that the bigger the molecule, the less possibility for her to penetrate into the adsorbent pore space, so, the bigger the molecule the less it will be retained.

INSTRUMENTATION HPLC instrumentation includes a pump, injector, column, detector and recorder or data system, connected as shown in the Fig. 13 below. The heart of the system is the column