Chapter 13 Evaporative Light Scattering Detection of Carbohydrates in HPLC

515

CHAPTER 13

Evaporative Light Scattering Detection of Carbohydrates in HPLC M. DREUX and M. LAFOSSE Laboratoire de Chimie Bioorganique et Analytique, LCBA UA 499, UniversitC d’orlians, B.R 6759, 45067 OrlCans Cedex 2, France

13.1 INTRODUCTION

Evaporative light scattering (ELS) detection is a recent detection technique [ 11 increasingly used in high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) of various compounds including carbohydrates. ELS detection acts as a qualitative or quantitative visualisation of each separated solute only if it is in a liquid or a solid state after the vaporization process. This detection principle is limited to solutes of low volatility, that is to say difficult to analyze using GC. Therefore, the main area of application of ELS detection is the field of HPLC. Which carbohydrate characteristics enable visualization (qualitative analysis) and determination (quantitative analysis)? Among specific properties, electrochemical and chiral activities are used and described in two different chapters (i.e. Chapters 10 and 14, respectively) whilst the chemical or biochemical reactivity of carbohydrates is described in two other chapters (i.e. Chapters 15 and 16, respectively). Other specific properties are more difficult to exploit: UV spectrophotometry, the most popular detection technique used in HPLC suffers from the lack of a suitable chromophore in the sugar molecule, as well as from convenient and constant sensitivity [2,3]. Also, the lack of a fluorophore group or an ionic group eliminates direct fluorescence or conductivity detection, respectively. Concerning non-specific properties however, different detection techniques are available. Mass spectrometry uses various ionization techniques that are discussed in Chapter 12 and refractive index detection, described in Chapter 11, enables the measurement either of a reflected or transmitted (deviation angle) energy or of the celerity of a light beam (interferometric) between two cells that contain the pure mobile phase (reference cell) and the modified mobile phase (sample cell). Comparisons of RI detection and ELS detection are discussed later in order to highlight the advantages of each one and their complementarity.

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For over fifteen years, HPLC has been the method of choice for the analysis of sugars due to the increased demand for selectivity, rapidity of separation and detection sensitivity. As is well known, however, the large number of isomers and homologous, the anomeric configurations and the instability in certain media (basic) make carbohydrates difficult to separate and determine. The presence of interfering products in complex media necessitates investigations into sample preparation and selection of the chromatographic system compatible with a suitable detection. The very complex nature of samples to be analyzed - carbohydrates are the most abundant family in natural products and are present in most food products - necessitates a sample pretreatment before injection into the chromatographic system. A review on the state of the art in sugar analysis by HPLC has been recently published by an author working in our research team [4] in which numerous aspects were considered. In this chapter, only the specific aspects of properties and performance of chromatographic systems and the associated ELSD are illustrated and developed. Aspects concerning sample preparation (see Chapter 1) and analytical and preparative separation (see Chapter 9) are very well documented in the present book.

13.2 EVAPORATIVE LIGHT SCATTERING DETECTOR DETECTOR TECHNOLOGY AND CHARACTERISTICS

- PRINCIPLE,

HPLC has developed extremely rapidly over the last fifteen years and significant advances have been made in all areas of instrumentation. Improvement in detection sensitivity was a great necessity, particularly in universal detection. Despite considerable practical improvements (automatic cell refill and auto zero) and improved performance (limit of detection) in the most commonly used universal HPLC detection, refractive index detection, a new universal detector was created from Charlesworth’s experiments [l] entitled an “evaporative analyzer as a mass detector for liquid chromatography”. The evaporator light scattering principle appears ideally suited when there is a large difference in volatility between eluent and sample. This principle excludes the analysis of volatile solutes, for which gas chromatography and suitable detection were conceived. The ELS detection principle is ideal for HPLC as well as SFC [5], high counter current chromatography (HCCC) [6] and field flow fractionation (FFF) [7]. Charlesworth’s work has opened the way for evaporative light scattering detectors (ELSD) that measure the scattered light generated by microparticles transported by a gas flow and directed through a light beam in order to scatter the light. Three different instrument technologies are currently marketed through the ACS [8], VAREX [9] and SEDEX [lo] models, the latter being the result of our laboratory’s research. The principle of ELSD is to nebulize the column effluent into droplets which are carried by a nebulizing gas (air, nitrogen, etc.) in an evaporator (or drift) tube and then directed towards a light beam. Light is scattered by residual particles of

Evaporative Lighi Scattering Detection of Carbohydrates in HPLC

517

MOBILE PHASE

1

I I

NEBULISATION

EVAPORATION

Fig. 13.1. Schematic of the ELSD.

non-volatile material and measured by a photomultiplier or a photodiode at an angle of 90" or 120" to the light beam source direction. The intensity signal is related to the solute concentration in the eluent and allows determination. As show in Fig. 13.1, three main operations are involved in the detector: (i) the nebulization (or atomization) of the mobile phase of the HPLC experiment which is transformed into a cloud of droplets, (ii) the vaporization of the cloud of droplets, and (iii) the scattering of the incident light by the cloud of residual particles. 13.2.1 Nebulization

All commercial detectors involve nebulization of chromatographic effluent into a gas stream with a Venturi nebulizer which is a pneumatic nebulization system. Only nebulizers with concentric flows (liquid to nebulize and gas for the nebulization process) are marketed even though the advantage of a cross-flow design for the limit of detection has been reported [ll]. During the second operation phase a divided spray is more easily vaporized than a bulk liquid. Divided spray generates residual non-volatile particles which scatter the light and determine the signal intensity. A constant nebulization process is needed for a satisfactory repeatability of analysis. This can be achieved in two different ways: either the whole aerosol is directed towards the evaporator tube for the generation of particles that produce the scattered light or only part of the aerosol is directed towards the evaporator tube. The ACS technology is the only one in which the whole aerosol produces the scattered light. In the VAREX technology, if the mobile phase flow rate exceeds a maximum value that depends on the mobile phase volatility, an adjustable splitter regulates the liquid stream towards the nebulizer. This option seems to be better for detection characteristics (linear dynamic range, limit of sensitivity) than increasing References pp. 539-540

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Chapter 13

in the evaporator tube temperature. In the SEDEX technology, part of the aerosol is selected by a nebulization chamber in which variable condensation on the walls limits the aerosol transport to smaller droplets. The use of a splitter before the nebulizer is restricted to preparative chromatography in which the goal is to recover the solute. The nebulization chamber comprises a waste pipe for the condensates. Discrimination in the droplet size changes the aerosol distribution which becomes narrower - bigger droplets condense more than smaller ones. Condensation during the second operation (vaporization step) is avoided or diminished and the contamination of the evaporating tube is decreased or eliminated. Moreover, the biggest droplets are eliminated and consequently lower vaporization temperatures are required for droplet vaporization. A low temperature is always more conducive to the formation of solid particles which scatter the light more than liquid particles of the same diameter [12]. In the case of solvents of relatively low volatility such as water, the SEDEX instrument is able to vaporize at lower temperatures than competing models. The SEDEX technology seems to be much better adapted than the others for reversed phase chromatography, elution with plain water and solutes that are thermosensitive. Several factors influence the average diameter of droplets and the droplet distribution. The average droplet diameter DO produced by the nebulizer has been approximated by Nukiyama and Tanasawa [13] and used by Mourey and Oppenheimer [ 141 (13.1) CTI, P I , ,UI are the liquid surface tension, density and viscosity, respectively, ug - UI is the difference between nebulizer gas and liquid velocities, and Q1/Qg is

where

the ratio of liquid to gas volumetric flow rates. Mourey and Oppenheimer [14] Guiochon et al. [15], Righezza and Guiochon [ll], and Van Der Meeren and Vanderdeelen [16] have demonstrated that the variation in droplet diameter for various organic solvents was not large at given liquid and gas flow rates. Moreover, the first term of Eq. (13.1) is seven to ten times greater than the second term [ l l ] and does not vary greatly as long as the gas velocities are large in comparison with liquid velocities [14]. In these conditions, nebulization temperature variation and gradient elution do not involve large variations in DO [14]. In conclusion, Do vary in the micron range from 4 to 40. With an increase in the gas flow rate, the average droplet diameter decreases and the detector response decreases accordingly. At low gas flow rate, the nebulizer does not function properly and spikes appear. For each instrument, a maximum response is observed with gas flow rate [14] and is dependent on nebulizer characteristics. Note that the recommended gas flow rate is around 15 l/min with the ACS instrument and 4-5 l/min with SEDEX and VAREX instruments. Each nebulization process has a characteristic distribution in size and with the Venturi nebulizer this was large. Nevertheless, a narrow distribution size must be

Evaporative Light Scattering Detection of Carbohydratesin HPLC

519

mobile Dhase

circulating

liquid

drain Fig. 13.2. Schematic of the nebulizer and the nebulization chamber of the SEDEX-ELSD.

achieved [17] in order to increase analysis repeatability. This goal is partly reached in the nebulization chamber of the SEDEX instrument. Finally, a constant gas flow rate is recommended particularly with nebulizer designs for which an enormous change in detector response has been observed [17]. Figure 13.2 illustrates details of the special nebulization system (nebulizer nebulization chamber) of the SEDEX instrument.

+

13.2.2 Vaporization

The vaporization operation takes place in a heated evaporator tube (or cylinder) in order to produce particles of pure solutes. As the solvent evaporates, there is a reduction in droplet size: the resulting diameter D is related to Do [ 14,151 by the following equation (13.2) where C and p are the concentration and the density of the solute, respectively. Calculations give a reduction in droplet size at a density of one unit by a factor of about ten at a solute concentration of 1000 ppm, and a reduction of about one hundred at a solute concentration of 1 ppm. Consequently, from the Do values comprised between 4 to 40 p m the scattering particles have a diameter D between 40 to 400 nm at 1 ppm and 400 nm to 4000 nm at 1000 ppm. These calculations do not take into account changes in droplet size distribution, which are caused by loss through condensation on the walls of the evaporator tube and/or by coagulation process [18]. Another point concerns the vaporization time; Charlesworth [11 calculated the time t d required for the droplet of solvent to be completely vaporized (13.3)

References pp. 539-540

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Chapter 13

where AHVIM is the molar volatility, p is the liquid density, k~ is the thermal conductivity of the gas film surrounding the particles and AT is the difference in temperature between the gas and the surface droplet. From Eq. (13.3) one can conclude that vaporization is time consuming for: (i) high particle size (DO)and high concentration [see Eq. (13.2)], (ii) low molar volatility ( A H v / M ) ,and (iii) gas of nebulization with a low thermal conductivity ( k f ) at a temperature very close to that of the eluent ( A T ) . The aim of vaporization is to eliminate the solvent while avoiding partial vaporization of the solute. ACS and SEDEX work with a room temperature gas stream while VAREX utilizes a preheated gas to increase A T . ACS and SEDEX have chosen a long tube length while VAREX a very short one. Which is preferable? The use of a long evaporator tube is an alternative solution to achieve complete vaporization at a high flow rate of mobile phase [17], but VAREX prefers using a stream splitter to evaporate a small quantity of eluent at a higher temperature. Increasing the heat causes solutes which possess a moderate or high molar volatility to evaporate, and consequently the scattered light has lower intensity. ACS and particularly SEDEX use a longer length for evaporation at moderate temperature, so their use is more widespread. The vaporization system in ACS uses more drastic conditions than in the SEDEX instrument because the cylinder gauze heater is in a direct contact with the solute. In SEDEX the tube is heated by an outside element that permits homogeneous temperature and reduces the degradation of thermosensitive solutes. This is an advantage in the case of some sugars [19] for which a low evaporator tube temperature is better than a high temperature. Comparison of the temperature adjustment of the different instruments is not easy because it does not reflect the solute particle temperature in the gas phase. The only representative measurement of the temperature of the gas stream is at the outlet of an instrument, but as this information is not available, there is no sense in comparing the vaporization temperatures used for the different instruments. Nevertheless, the lower the temperature of gas and solute, the lower is the photomultiplier noise and the easier it becomes to reach the preferential state of the solute, namely a solid state [12]. The advantages of helium (high value of kf)as a nebulization gas over the classical gases used (air, nitrogen, carbon dioxide) for both volatile, non-volatile and thermolabile analytes have been emphasized by Van Der Wal [18]. 13.2.3 Measurement of the scattered light intensity

- quantitative analysis

The particle cloud emerging from the evaporator tube is passed through a light beam and the amount of light scattered by the particles is measured by a photomultiplier or a photodiode at a fixed scattering angle. The emission source, in the visible region of the spectrum (400 to 800 nm), is multiwavelength in the ACS and SEDEX models, while a monowavelength laser operating at 670 nm is used by VAREX. Consequently, VAREX does not function properly for solutes which possess a chromophore group at the wavelength of the laser [ l l ] .

Evaporative Light Scaitenng Detection of Carbohydratesin HPLC 2

1

67 nm

I A= 500ni

'/////,A

1PPm 50nm

2 PPm

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3

521 loq0

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I

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1000 P P m 1 10000PPm 5000nmh\\\\\

2000 ppm

I

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The influence of the scattering angle has been demonstrated and there is no ideal or optimal angle [20]. Relatively to the source beam direction, ACS uses an angle of 45", VAREX an angle of 90" and SEDEX an angle of 120". In order to protect the optical part of the instrument, SEDEX adds to the inner particle stream an outer gas stream which envelops the first one; this gas guide protects the optical part from solvent vapor mixed with the nebulization gas and avoids spikes. A good efficiency of the guide is obtained if the speed of the outer gas is higher than the speed of the inner particle cloud gas. ACS and VAREX avoid exposing the photomultiplier to the eluent vapor by using a fibre optic cable system in order to transmit and measure the scattered light. The mechanism of the light scattering by particles is complex and may result from different contributions. Their relative importance depends on the value of ratio of the scattering particle diameter D to the wavelength h of the incident source beam. At a constant h value (e.g., laser source) with small particles such the D / h < 0.1, the scattered light is in the Rayleigh region. At 670 nm it necessitates D < 67 nm. As the particles become larger (0.1 < D / h < 10) it is the Mie scattering, while beyond D / h > 10 the classical theory of reflection-refraction applies [I]. Clearly in order to decide which mechanism is responsible for the scattered light measured, D calculations associated with a concentration range afford an estimation. Figure 13.3a illustrates that at low concentration (about a few pprn and less), the amount of scattered light comes from Mie and Rayleigh domains, and at high concentration (more than 1000 ppm) Mie and reflection-refraction contribute to References pp. 539-540

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Chapter 13

the total scattered light measured. In the Rayleigh domain, the scattered light is related to the D / h ratio at power 6, while in the Mie domain the power is 4 and 2 for the reflection-refraction domain [21]. Besides, a particle acts as an individual point source or as a multiple point source [l] relative to its diameter. In conclusion, it is not surprising to find that a calibration curve of solute concentration against detector response is sigmoidal [1,14,18]. Although the variation of the response is complex (it depends on droplet size, concentration and nature, gas and liquid flow rates, vaporization temperature etc.), it was assumed that in a large range of sample size the measured peak area can be related to sample size by the following relationship A=amb

(13.4)

where b is the slope of the response line, m is the mass of compound injected and a is the response factor. As a result, plots of the peak area versus the mass (or concentration) in double logarithmic coordinates are linear with a slope b logA = blogm + l o g a

(13.5)

The limit of linearity is beyond three decades in concentration in order to describe only the Mie domain (see Fig. 13.3b). Consequently, in the intensity function I = f(D/A)4, the replacement of D by the concentration [Eq. (13.3)] gives 1 = f’(C)4/3

which yields for slope b a value of 4/3 = 1.33. A slope higher than 1.33 characterizes the Rayleigh domain [(C)6/3= C2]and a slope b lower than 1.33 characterizes the reflection-refraction region [ c ~ / ~ ] . Figure 13.4 shows that at high concentration a decrease in slope b occurs while at low concentration a higher value of slope b is obtained. Assuming a constant background noise, the detector response a is higher in the reflection-refraction domain than in the Rayleigh domain. Each deviation in slope and response is the expression of parameters that have not been taken into account by Eq. (13.3), such as partial vaporization of the solute, loss on the wall of the vaporization tube, and coagulation [18]. Righezza and Guiochon [ l l ] and Mengerink et al. [18] reported an increase in coagulation phenomenon with a decrease in the gas flow rate. Recent experiments on nucleation [22] demonstrated the importance of coagulation on a scattering system - ppb concentration can be detected - and reinforces our own observations at low concentration of some solute ( < 1 ppm) in the presence of ethanol in the eluent. The linear dynamic range of an instrument is dependent on numerous parameters and in double logarithmic coordinates it spreads over two or three decades. The lowest response corresponds to the widest dynamic range and the slope of the calibration curve is increased with more volatile compounds [1,18]. So at low concentration it is very important to use a low temperature in order to favor the

Evaporative Light Scattering Detection of Carboh-ydratesin HPLC

523

I;;I . I

14. Raleiqhi

: 1

Reflection Refraction

,

I

I

1 .l

3.7

LoqO ( 0 in pm) Fig. 13.4. Variation of the slopes of calibration curves. Calibration curve equations in logarithmic coordinates are deduced from the scattered light intensity I = f ( D / A ) ' = f ' ( C / A ) X / 3where , x = 2, 4, 6 in the reflection-refraction, Mie and Rayleigh scattering, respectively.

formation of larger droplets, low solute vaporization and crystallization. To increase the linear dynamic range towards the high concentrations, Mourey [20] suggests decreasing the intensity of the emission source. The slopes mentioned in the literature have values generally comprised between 1 and 1.6, with 1.3 being the most representative value. It has been observed by different authors that inside a group of homologous compounds the detector response is nearly equal [12,23-251, so Asmus and Landis [26] concluded that ELSD is suitable for performing the assay of impurities in drug materials to ensure quality in products. Concerning the limit of detection, concentrations as low as ppm or less have been reported but often in direct injection (without column) or by extrapolation. Specifications given by manufacturers spread over one nanogram to ten nanograms, which corresponds to 20 p1 injected volume of 50 ppb to 500 ppb of a solute solution. 13.2.4 Characteristic properties of the detector

ELSD is very easy to use and is ready to function only a few minutes after power is turned on. A very low background signal is observed since there is no light scattered by the solvent vapors or by the nebulization gas. The use of solvents with low dry solid residue is a more important characteristic

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Chapter I3

of the solvent than its UV transparency. Filtration of the solvent to remove suspended particles is advised and the solvent quality required for preparative chromatography is often better adapted to ELSD requirements than HPLC quality solvents. The presence of a stabilizer in any solvent will lead to no loss in limit of detection as long as its volatility is lower than that of the eluent. Gradient elution produces baseline drift only when each of the solvents to be mixed produces a different response. The repeatability of the detector response is best achieved when the operating conditions guarantee a constant flow rates of nebulization gas and eluent, a constant vaporization temperature and no partial vaporization of the solute. In the SEDEX model, nebulization at a low controlled temperature has been performed and it can be chosen independently of the vaporization tube temperature. The relative standard deviation of the signal is often more than 1%, that is to say higher than with UV detection; the dynamic nebulization process induces this drawback. The linearity of the response enables determination in a concentration range of two or three decades and a calibration curve is always required. Nevertheless, a response practically independent of the compound is obtained in different homologous series as long as the same physical state (liquid, solid, etc.) is encountered. Attention must be paid to chromatographic systems that use a low vaporization temperature and afford crystallization and/or constant response. Asmus [26] in a steroid series has found a specific chromatographic system for quantitative determination. ELSD is not a true mass detector. For calculations in double logarithmic coordinates, VAREX offers a specific supplementary instrument and SEDEX a specific additive program used with some models of Shimadzu calculators. In the case of interfering peaks, Guiochon and co-workers [17] have demonstrated that the resolution given by the ELSD appears to be better than it really is. This result is interesting in the case of a coupling of ELSD with another detection. As a second detection, the decrease in the resolution with ELSD becomes negligible, and nearly equal to the resolution observed with the first on line detection. On line coupling of two detections is always fruitful for the chromatographic knowledge of a complex analysis. Specific UV detection on line with universal (ELSD) detection has demonstrated interest in the case of a mixture of UV- and non-UV-absorbing compounds [27]. ELSD functions with a Venturi nebulizer which creates a depression. In these conditions, RID can act on line with ELSD without the risk of RID deterioration. Finally, cleaning ELSD is easy but the process differs considerably between the various instruments. 13.2.5 Which detector should be used with a chromatographic analysis of a given mixture? If the solutes are characterized by a lower volatility than the eluent used for chromatographic analysis, ELSD is a good choice. If the solutes do not

Evaporative Light Scattering Detection of Carbohydratesin HPLC

525

possess chromophore or fluorophore and are present in a complex matrix, ELSD becomes the best choice and perhaps the only choice since a gradient elution is needed. As information on the volatility of a solute is not always easy to come by, the following scheme must be used in order to test ELSD compatibility with the solute to be analyzed: (i) Prepare solute solutions at two or three different concentrations in solvents used for chromatographic analysis, after proving no response of these solvents with ELSD; (ii) Determine the minimum vaporization temperature for a total vaporization of eluent which leads to the lowest partial vaporization of solute; (iii) Without column, observe an increase in ELSD response with the different concentrations of solute solutions - comparison with the response of well known solutes (test solutes) provides useful information; (iv) Check your chromatographic system without and with column. The eluent must provide a constant background noise. If a difference is observed high sensitivity or gradient elution are more difficult to carry out. 13.3 ANALYSIS OF CARBOHYDRATES AND CARBOHYDRATE DERIVATIVES 13.3.1 Separation-detection dependency

13.3.1.I Eluents incompatible with ELSD characteristics ELSD requires the vaporization of the components of the eluent and the nonvaporization of solutes in the heated evaporator tube. It is thus possible to use any gradient requiring the lowest possible temperature to prevent thermal degradation of the sugars. Therefore, the chromatographic eluent is not independent of the detection system. ELSD cannot be used, however, to separate sugars on “in situ” amine impregnated silica gel (see Chapter 3) where the mobile phase contains a non-volatile modifier which has two amine functions to interact both with the solute and with the silica gel [28-301. Similarly, ELSD cannot be used to monitor the separation of sugars on copper silica gel, where the mobile phase is a mixture of acetonitrile-aqueous copper solution [31]. Furthermore, oligosaccharides separation by anion-exchange chromatography with an aqueous sodium hydroxide and sodium acetate eluent [32,33], which has been developed for pulsed amperometric detection (Chapter lo), are also incompatible with the principle of ELSD. Assays to change sodium hydroxide by ammonia can afford compatibility with ELSD but the resulting selectivity of the separation is questionable. 13.3.1.2 Volatile eluents Simple carbohydrates (i.e. saccharides and oligosaccharides) are more soluble in water than in organic solvent and therefore water is always a constituent of the References pp. 539-540

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Chapter 13

mobile phase. Water has a low volatility, consequently the SEDEX 45 ELSD is better adapted than the other types of detectors to water vaporization: the required evaporator tube temperature needs only be 40-50°C to obtain total vaporization of water flow rates as high as 2-3 ml/min. Heating to high temperatures is a drawback because the detector response depends on the vaporization temperature: pentoses and fructose show a marked decrease in response at elevated temperatures [ 191. Sucrose degradation was also noted when the vaporization temperature setting is about 70°C with the ACS model 750/14 [34].

13.3.1.3 ELSD quality of a chromatographic eluent A high background noise has been observed with certain qualities of solvents [35]. This phenomenon is not related solely to the level of dry residue, and remains unexplained. To assess the ELSD quality of a chromatographic solvent, the method consists in connecting directly the column to the detector: the background noise must be low and similar to the one obtained when the pump is stopped. 13.3.1.4 Stability of stationalyphase The lack of stability of the stationary phase with regard to the eluent can disturb the detection by an increase in the background noise and by a baseline drift. This has been noted when an acetonitrile-water mixture is flushed through an aminopropyl silica packing [ 19,361. As shown in Fig. 13.5a, when the water content of the mobile phase reaches 30-40% (v/v), the baseline drifts and the background noise increases considerably. The drift corresponds to an increase of scattered light due to non-volatile microparticles arising from the hydrolysis of aminopropyl silicas [35]: about 30 ppm silicium in the effluent has been titrated by atomic absorption. This drawback is similar with all commercialized aminopropyl silicas: consequently gradient elution at increasing water content becomes difficult and the sensitivity of the sugar detection by ELSD is lowered by about 1 pg. Consequently, analysis of an oligosaccharide mixture namely maltodextrin analysis reduces the quality of carbohydrate determination 119,361. Hydrolysis is not observed, however, with diol-bonded silica (Fig. 13.5a) so these packings can be used instead to the aminopropyl silicas with acetonitrile-water mobile phase. Figure 13.6 shows an excellent separation of several carbohydrates using a gradient elution with an acetonitrile-water mixture on Lichrosorb Diol. This chromatogram cannot be observed with a refractive index detector with which gradient elution can not be used. With polyol-bonded silica [37], an additive to acetonitrile-water is required to avoid the broadening of peaks due to the anomeric forms of the sugars. Addition of triethylamine is compatible with ELSD and requires only a slight increase in vaporization temperature. Note that this addition is precluded with UV detection. The analysis of monosaccharides and polyols shows a good selectivity on cationexchange resins (Ca2+ for example), using pure water at 80-90°C (see Chapter 4).

Evaporative Light Scattering Detection of Carbohydrates in HPLC

527

Fig. 13.5. (a) Baseline drift with aminopropyl and diol bonded silicas and gradient elution from A (plain acetonitrile) to W (plain water). I = Lichrosorb NH2; 2 = Nucleosil OH; 3 = Zorbax OH; 4 = Lichrosorb Diol, Zorbax ODS, Lichrospher Diol. (Reproduced from Ref. 35 with permission of Elsevier Science Publishers.) (b) Baseline drift with Ca*+-exchanger resin and water elution during an increase of the evaporator tube temperature. (Reproduced from Ref. 38 with permission of Elsevier Science Publishers.)

0 5 10 15 20 25min Fig. 13.6. Analysis of sugars mixture by gradient elution. Column: Lichrospher Diol, 250 x 7 mm. Eluent: acetonitrile (A), water (B). 1 = fructose; 2 = glucose; 3 = sucrose; 4 = lactose; 5 = raffinose; 6 = dextrans. (Reproduced from Ref. 35 with permission of Elsevier Science Publishers.)

At room temperature the background noise and baseline are similar to those obtained with pure water on a c18 column [38]. This means that only a low release of ions occurs during elution with pure water. When the column temperature References pp. 539-540

528

Chapter 13

reaches 80-90°C, the amount of scattered light increases as does the background noise. Figure 13.5b [38] illustrates a permanent release of calcium salts which gives a very constant low background noise. However, the signal to noise ratio is similar to the one given by the octadecyl-water system [35] because the cationic resin-water system causes an increase in signal (a sugar-calcium complex increases the detector response) and an increase in noise at the temperature of the analysis. After a large elution volume of water, SO;-Ca2+ groups of resins are transformed into acid groups SO;H+ and carbohydrates are hydrolized during their analysis at 80-90°C [35]. Regeneration of the chromatographic system should be performed by flushing a solution of calcium nitrate or acetate. Control of this regeneration can be monitored by ELSD with injection of sugar which produces the by-products of hydrolysis. The sensitivity of ELSD enables regeneration to be easily and efficiently controlled. 13.3.1.5 Automatic sugar analysis in a complex mixture ELSD offers additional advantages with regard to refractive index detection for automatic analysis. In fact, ELSD shows (i) no lack of performance with flowrate variation and/or column temperature fluctuation, (ii) rapid equilibration, and (iii) no zero drift. These advantages enable easy determination of carbohydrates (glucose, fructose, sucrose and raffinose) in molasses (Fig 13.7a) with an on-line purification of raw products [39,40]. A sample clean-up is carried out on an automatic sample processor injector (ASP1 model 232-401 Gilson). The stationary phase was a Zorbax ODS preceded by a precolumn permitting the adsorption of the polysaccharides and other molecules included in the molasses. The precolumn is cleaned by flushing with methanol and then water during the elution time of carbohydrates on the analytical column (Fig. 13.7b). The rotation of valves during cleaning is compatible with ELSD while the rupture of flow disturbs the RID baseline too much. 13.3.2 Isocratic and gradient HPLC with polar and apolar stationary phases 13.3.2.1 Diol and polyol stationary phases We have noted the advantages of diol stationary phases relatively to the aminopropyl silica with acetonitrile-water mobile phases. However, because a low percentage of water in the eluent is necessary to achieve good selectivity of monoand disaccharides, it may cause some solubility problems for certain sugars such as lactose. Since diol columns offer excellent stability with no Schiff’s base formation of reducing sugars, we have explored new eluent systems. We previously obtained good results [41,42] by SFC and more specifically by using Lichrospher and Lichrosorb Diol silica gels and a C02-methanol eluent (Fig. 13.8a). As the polarity of this supercritical fluid mixture is nearly equal to that of a dichloromethane-methanol mixture, we have therefore tried to transpose the system in HPLC comparatively

Evaporative Light Scattering Detection of Carbohydrates in HPLC

529

a

C R

Fig. 13.7. (a) Schematic of automatic injection and sample clean-up instrumentation. PC = precolumn; C = ODS-silica column; V1 = injection valve; V2 = switching valve; S2 = cleaning solvent (water); S3 = cleaning solvent (methanol); D = ELSD. (Reproduced from Ref. 40 with permission of Hiithig Publisher.) (b) Chromatogram of molasses without purification. Column: Zorbax ODS (250 x 4.6 mm). Eluent: water. Solutes: I = inorganic ions; 2 = monosaccharides and organic acids; 3 = sucrose; 4 = raffinose; 5 = betaine and unknown. ELSD: evaporator tube temperature, 40°C. (Reproduced from Ref. 39 with permission of Hiithig Publisher.) (c) Chromatogram of purified molasses. Solutes: G = glucose; F = fructose; M = maltose (internal standard); S = sucrose; R = raffinose. (Reproduced from Ref. 39 with permission of Hiithig Publisher.)

References pp. 539-540

Chapter I3

530 rnE11Rh

8%

n

0

lorn,"

Fig. 13.8. Chromatograms of sugars in SFC (a) and LC (b) on Lichrospher Diol (250 x 4 mm) 5pm. (a) Eluent: COz-MeOH, 84.5 : 15.5; flow rate: 1.8 ml/min; pressure: 270 bars. ELSD: evaporator tube temperature, 45°C. (Reproduced from Ref. 42 with permission of Elsevier Science Publishers.) (b) Eluent: dichloromethane (A)-methanol (B). Solutes: dR = deoxyribose; mE = meso-erythritol; Rh = rhamnose; X = xylose; F = fructose; M = mannose; G = glucose; S = sucrose; M e = melibiose; R = raffinose. ELSD: evaporator tube temperature, 40°C. (Reproduced from Ref. 43 with permission of Vieweg Publisher.)

with other polar columns: Zorbax NH2, RSil NO2 and Zorbax TMS [43]. These various polar columns afforded complementary selectivity and have been used to analyze sugars in glucose syrup, tobacco or beet juice. As has been demonstrated [43], careful attention must be paid to the compatibility between injection solvent and mobile phase composition. With these organic eluents, a gradient elution permits a separation of mono-, di- and trisaccharides (Fig. 13.8b) without baseline drift [43] and with a good sensitivity (20 ng). To compare the mechanism of retention of sugars and polyols in SFC and LC, chemometric studies were investigated [44]. Automatic classification and factor analysis methods have shown the homogeneity of retention mechanisms. Principal component analysis allows more interesting investigations: the retention of sugars and polyols is only the sum of two mathematically independent mechanisms. MorinAllory and Herbreteau [45] have demonstrated that the major part of the retention is directly linked to some specific hydroxyl groups included in the sugar molecule. The results on diol columns with dichloromethane-methanol mixtures have been transposed on bare silica gel [46]. With a low water content in the eluent (0.2%) to deactivate the silica surface, reproducible results were obtained. Various studies have shown that due to a partition phenomenon the retention increases with increasing the surface area of silica and increasing the water content in eluent (0-1%). On the other hand, retention decreases if dichloromethane is replaced by chloroform.

Evaporative Light Scattering Detection of Carbohydrates in HPLC

53 1

limin

Fig. 13.9. Separation of mono-, di- and trisaccharides by gradient elution on Zorbax Sil (250 x 4.6 mm). Gradient elution: dichloromethane-methanol-water (80 : 19.8 :0.2) during 5 min, then 45 : 54.8:0.2 in 2 min. Solutes: see Fig. 13.8. ELSD: evaporator tube temperature, 40°C. (Reproduced from Ref. 46 with permission of Elsevier Science Publishers.)

In order to obtain reproducible results, a simple method is proposed to equilibrate silica gel by flushing the column with dichloromethane before elution. With this method a reproducible gradient elution is possible and permits a rapid separation of mono-, di- and trisaccharides (Fig. 13.9). In conclusion, diol systems can be extremely useful but the solubility of sugars limits their use to mixtures having a low sugar concentration [43,46].

13.3.2.2 Aminopropyl-bonded silicas We have previously noted (Section 13.3.1.4) that the hydrolysis of the aminopropyl silicas increases when the water content of the mobile phase reaches 30-40%. To improve the use of these packings, ternary mixture with low water content and acceptable elution power has been investigated. An example of the use of an acetonitrilemethanol-water (30 : 59.5 : 10.5) mixture to analyze oligosaccharides contained in a maltodextrin is shown in Fig. 13.10a. Maltodextrins are eluted more rapidly than with a binary acetonitrile-water 70: 30 eluent without (Fig. 13.10b) the hydrolysis of bonded silica and the observation of baseline drift and background noise. Ternary mixtures as eluent afford new possibilities for using aminopropyl silicas but only for oligosaccharide determination (two to less than ten monosaccharide units per molecule). Indeed, the polysaccharides (ten or more monosaccharide units) are strongly retained on these columns and cannot be eluted even with a greater eluting power of a methanol-water mixture with high water content as we demonstrate later. It is preferable to use another method [38] to elute oligo and polysaccharides easily (see next section). Another efficient method to analyze saccharides and oligosaccharides with amino stationary phase consists of using References pp. 539-540

Chapter 13

532

lkmin

lkmin

Fig. 13.10. Analysis of maltodextrins. Column: Erbasil NH2 (250 x 4.6 mm); flow rate: 1 rnl/min. (a) Mobile phase: acetonitrile-methanol-water (30 : 59.5 : 10.5). (b) Mobile phase: acetonitrile-water (70: 30). ELSD: evaporator tube temperature, 45°C.

a new packing of Asahipak (Asahipak NH2 P-50) based on NHz-bonded vinylic alcohol copolymer. This packing does not hydrolyze with the high water content and no background noise is observed. Capabilities are under investigations.

13.3.2.3 Apolar stationary phases Octadecyl-silica-bondedphases with pure water as eluent is an easy method for the analysis of mono-, di-, and trisaccharide mixtures, but it affords a poor resolution in the monosaccharide family and permits the separation of oligosaccharides only. As discussed in Chapter 2, peaks of high-dp oligosaccharides are broad while polysaccharides are not eluted in pure water. Consequently, elution of this kind of polymers requires a good solvent [47]. Here addition of methanol to the eluent was chosen [38]. So a gradient elution at increasing methanol content in water permits an excellent analysis of oligosaccharides and polysaccharides (Fig. 13.11). We have added to this analysis the chromatogram of B-cyclodextrin showing that this compound which has 7 monosaccharide units per cycle has a more hydrophobic character than the corresponding linear one in maltodextrins (7 glucose units). /I-Cyclodextrin is eluted close to polysaccharides contained in maltodextrins. In contrast, cyclodextrin is eluted near stachyose (4 units of monosaccharides) on polar packing [48], showing a weaker polarity than the corresponding non-cyclic sugar. Cyclodextrin shows more hydrophobic interaction than linear sugar in reversed phase chromatography - behavior confirmed by the retention on Hypercarb [49] - and less polar interaction in polar partition chromatography.

13.3.2.4 Quantitative determination Table 13.1 compares the response of sugars with ELSD, UV detector, RID and

PAD.The last three do not respond uniformly to all sugars and calibration curve for each sugar must be determined. Indeed, with UV detector the response of fructose is 200 times greater than that of maltose, which explains the abnormal relative

Evaporative Light Scattering Detection of Carbohydrates in HPLC

1

533

P

J 0

lbmin Fig. 13.11. Chromatogram of maltodextrins by gradient elution. Column: Lichrospher 100 RP 18 end capped (100 x 4 mm), 5 p m . Eluent: methanol-water; water during 5 min, followed by gradient elution from 0% to 80% methanol in 8 min, then 80% methanol. Solutes: G = glucose; M = maltose; 0 = oligosaccharides; P = polysaccharides, CD = cyclodextrin. ELSD: evaporator tube temperature, 45°C. TABLE 13.1 MASS RESPONSE FACTORS * OF SACCHARIDES WITH RESPECT TO GLUCOSE Detector

uv

Refractometry

Solute

ELSD

(8)

(a)

(b)

Pulsed amperometry (b)

Quantity injected (wg) 18

18

8.6-38.6

0.4-3.8

20 1.07(c) 1.12(d) 1.10(d) 1.00 (d) 0.94 (d) 1.15(d) 1.03(c) 0.97 (d) 1.09(d) 0.97 (c)

192 nm

Ribose Xylose Fructose Glucose Galactose Sucrose

1.76 1.13 4.30 1.oo 1.01 0.95

0.43 0.81 0.97 1.00 0.48 1.05

0.68 0.94

0.75 1.oo 0.97 1.07

0.73 1.06 0.74 1.oo 1.09 0.23

Lactose Maltose

0.54 0.02

0.79 0.12

0.92 0.53

0.63 0.43

RSD (%)

97

46

21

41

7

* Mass response factor is the relative response of each saccharide for a given quantity. (a) Mobile phase = acetonitrile-water 80:20 (v/v) [3]. (b) Mobile phase = aqueous eluent containing 0.1 M NaOH [58]. (c) Mobile phase = water. (d) Mobile phase = acetonitrile-water 80: 20 (v/v).

References pp. 539-540

Chapter 13

534

cl

0.07

4

z

3

=fl

RID

0.05

c

Y

ki

0.02

-0.01

ELS0

I

I

I

I

I

I

1

0

5

10

15

20

25

30

TIME (minutes)

Fig. 13.12. Comparison between the RID and the ELSD chromatograms of sugars. Column: Lichrosorb RP 18. Eluent: acetonitrile-water (80 :20), automatic pumps mixing. Solutes: SSP = sample solvent peak; I = rhamnose; 2 = xylose; 3 = fructose; 4 = glucose; 5 = sucrose; 6 = maltose; 7 = melibiose. ELSD: evaporator tube temperature, 40°C. (Reproduced from Ref. 50 with permission of Marcel Dekker, Inc.)

standard deviation (RSD 97%). With RID, the response of sucrose is about nine times that of maltose using an acetonitrile-water eluent and only twice as great with the second eluent. With PAD,the response of galactose is about five times that of sucrose and twice that of maltose. These differences also give a large RSD (20-40%). In contrast, ELSD response is nearly equal whatever the sugar molecule (RSD 7%). In addition, UV detector necessitates high quality solvents and can only be used in the case of non-complex sugar mixtures. RID requires premixing of the water and acetonitrile in order to increase the limit of detection and is sensitive to variations in flow and temperature. Clement [50] has demonstrated the advantage of ELSD over RID in isocratic elution (Fig. 13.12). In quantitative analysis, a calibration curve is obtained for glucose with water eluent on octadecyl-silica (Fig. 13.13). We can observe that the linearity between surface area response and concentration is better in double logarithmic coordinates than in simple coordinates because seven points on the eleven are superimposed in simple coordinates (Fig. 13.13a) while in double logarithmic coordinates these points are spaced (Fig. 13.13b). In this case the linear dynamic range is about 3 decades (concentration varies from 0.975 ppm to 1000 ppm). To compare the calibration curves of sugars, various sugars have been chromatographied with water or acetonitrile-water as eluents. Table 13.2 gives the b and a values of the parameters of equation (13.5). We note an average response factor of 1.24 (RSD = 3.55%) with water eluent and 1.09 (RSD = 4.8%) with

Evaporative Light Scattering Detection of Carbohydrates in HPLC

535

h e a r Regression

~

t

15ooOOO 12M)ooo

300000

0 600

300

0

1200

900

ConconhaRon (pprn)

-

Ln Ln Regression 15

T

-1

1

3

5

7

9

Ln Concentration (pprn)

Fig. 13.13. Calibration curves of glucose with water elution on Lichrospher RP 18 (100 x 4 mm), 5 pm. Concentration: 0.97 to 1000 ppm. r = 0.9985 in (a) and r = 0.9985 in (b). TABLE 13.2 SLOPE b AND INTERCEPT loga OF EQ. (13.5) RELATIVE TO THE CALIBRATION CURVE Eluent Acetonitrile-water 80 : 20 (v/v)

Eluent Water

Sugar

b

log a

Sugar

b

log a

Glucose Galactose Fructose Lactose Sucrose Maltose Average value

1.oo 1.11 1.08 1.16 1.10 1.09 1.09

8.54 7.71 8.0 7.40 7.96 8.00 7.93

Ribose Xylose Glucose Sucrose Maltose

1.30 1.19 1.20 1.24 1.25

5.0 6.1 5.9 5.7 5.6

Average value

1.24

5.7

RSD (96)

4.78

4.75

RSD (%)

3.55

7.35

536

Chapter 13

acetonitrile-water eluent. These values are slightly weaker than the ones obtained by Guiochon with methanol-water mixture [15] and which are the same (1.31) for glucose, fructose and maltose. Consequently, the difference between calculated and measured surface areas for a given concentration is about 2-20% with each eluent. So, for a given eluent only one calibration curve represents the response of every sugar; this result explains the name originally given to ELSD by ACS: the mass detector [8,19,51]. This approximation is partly true and ELSD is the one and correct trade name.

13.3.2.5 Carbohydrate derivatives Alkylglycosides [52] and alkylthioglycosides are carbohydrate derivatives used as biological surfactants and as solubilization and purification agents of membrane proteins. The characterization of their hydrophilic-lipophilic behavior has been demonstrated using chromatographic parameters such as k’ by reversed-phase liquid chromatography 1531. A comparison of methanol-water and acetonitrilewater mixtures has been done to improve separation according to the alkyl chain length, the polar head (sugar moiety) and the 0-or S-bonding between polar head and alkyl chain. Optimum linear gradient was obtained with acetonitrile-water (Fig. 13.14a). In a similar area of derivatives, several alkyl glucosinolates have been studied by RPC. Gradient elution with methanol-water eluent shows (Fig. 13.14b) a stable baseline whereas a drift is normally noted with UV detection at 234 nm. Saponins are steroids or triterpenoid glycosides having biological properties and are found in food plants. Because of the lack of UV chromophore, Ireland [54] has used ELSD with organic-acidic water eluent (1% acetic acid) and polar-bonded stationary phase. Becart [55] obtained successful separation with RPC and SEDEX 45 ELSD for Ginseng saponin extracts (Fig. 13.15) with a sensitivity of about 100 ng (S/N = 3). We underlined at the beginning of this part (13.2.1.1.) the difficulty in using ion-exchange chromatography with non-volatile eluent. To analyze organic acids such as tartaric or lactic acids, sulfuric acid is usually used as mobile phase on an Aminex resin. Replacement of sulfuric acid by the more volatile trifluoroacetic acid gives the same selectivity of analysis [56]. Other counter ions can be used on the anion-exchanger resins such as aqueous solution of acetic, formic acid or nitric acid. The use of a concentration gradient of nitric acid has enabled us to elute an inositol phosphate mixture without baseline drift and to determine its composition with accuracy and sensitivity [57]. 13.4 CONCLUSION AND FUTURE PROSPECTS

The ELSD has been proven useful for carbohydrate determination because of the low volatility of carbohydrates and their derivatives, and because of the possibility of using a gradient elution every time the complexity of the carbohydrate mixture

Evaporative Light Scattering Detection of Carbohydrates in HPLC

537

Fig. 13.14. Chromatograms of alkylglycosides (a) and alkylglucosinolates R-GI (b). (a) Column: Lichrospher RP 8 (100 x 4 mm), 5 p m . Linear gradient elution: acetonitrile-water from 25:75 to 55 : 45 i n 15 min. Peaks: 1 = Cg Glucose; 3 = Cx Galactose; 4 = CR Glucose; 5 = Cs Thioglucose; 7 = CH Xylose; 9 = C ~ O Maltose; I0 = Clo Glucose; 11 = Cl2 Maltose; 12 = Clz Glucose. (Reproduced from Ref. 53 with permission of Elsevier Science Publishers.) (b) Column: Lichrospher RP 18 (100 x 4 mm) 5p m. Linear gradient elution: methanol-water from 40 : 60 to 75 : 25 in 10 min, ~ 3 = Ctz GI; 4 = C I GI; ~ 5 = C14 GI. ELSD: evaporator then 75:25. Peaks: I = Clo GI; 2 = C IGI; tube temperature, 45°C.

requires it. The ELSD's sensitivity and principally its low detection limit depend on the machine's capacity and the separation methods used. Sensitivity can be improved by a better quality solvent (except in the case of water), and by increasing the amino column stability, eluent selectivity, and new chromatographic systems. Amino silica columns with ternary eluents using a low water content, a newly marketed polymeric amino column, can greatly improve the detection limit and gradient capabilities. SFC packed columns have been effective and the availability of efficient new systems (Gilson, Hewlett Packard) has increased their popularity. Complementary material to HPLC and SFC is obviously a bonus. Today on cyanobonded silica, only SFC gives selectivity for sugar analysis [42,56,59]. The solubility

References pp. 539-540

Chapter I3

538

& R'O

RO

1

a '

DerivmlYns 01 20 S Protopanaradlol

I

4

8 min

R

= D Glu D Glu

1 R' = D GIU D Glu

2 R' = L Ara D Glu (luranooe) 3 R' IL A n D GIU (pyranose)

4 R' ID GIv

12

Fig. 13.15. Ginseng saponins extract. Column: Lichrospher RP 18 (125 x 4 mm). Mobile phase: (A) = water + acetic acid (1%); (B) = acetonitrile %B/(A + B) from 20 to 40 in 15 min. Flow rate: 1 ml/min. ELSD: evaporator tube temperature, 50°C.

of highly polar and/or ionizable compounds in carbon dioxide, and polar modifier supercritical fluid mixtures, is limited but the compatibility of a very complex eluent composition with detector makes it possible to analyze non-derivatized amino acids

PI.

The development of ELSD in sugar analysis should be enlarged by using desalting systems. The limitation of additives to moderately volatile compounds can be partially carried out by using non-volatile salts and chemical suppression devices conceived for increasing the sensitivity for ion determination by conductivity detection [61]. Using a droplet size control and distribution device with ultrasonic nebulization and a nucleation control process, ppb determination becomes feasible [22]. Improvements in automatic concentration calculations and in the pollution and/or security aspects (recovery output of the detector of toxic eluent vapors diluted in the gas phase) should help convince the opponents of the ELSD of its usefulness. The ELSD's major advantages are that it is easy to use and ready to function after a few minutes warm-up, and it is on-line with diverse specific detectors as well as with the universal RID, and with various separation techniques (HPLC, SFC, HCCC, FFF). It appears to be the only inexpensive universal detector in packed

Evaporative Light Scattering Detection of Carbohydrates in HPLC

539

column SFC currently being used today, and should prove to be indispensable as a universal detector in HPLC in the future. 13.5 REFERENCES 1

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