Quantitative Chromatographic Analysis

Thomas Beesley, Benjamin Buglio

• Publisher: CRC Press
• Year: 2000
• ISBN:
  9781135553500
• Edition: 1
• Topic: Physical Sciences
• Subtopic: Chemistry
• Index: Chemistry

Highlights critical factors involved in successful chromatographic analysis. Details analytical procedures; outlining sample preparation, collection, transportation, and storage. Provides step-by-step guidelines for producing analytical reports.

Part 1
Introduction to Quantitative Chromatographic Analysis

Chapter 1
The Critical Factors that Govern a Successful Quantitative Chromatographic Analysis

Historical Introduction

The early work in liquid chromatography from Tswett onward involved almost no quantitative assays, but was largely used as a preparative technique, quantitative evaluations being carried out offline using separate analytical methods. Actual quantitative assays made directly by monitoring the column eluent commenced with gas chromatography (GC), first used in this way by the inventors of the technique, James and Martin, in 1952 [1] for the analysis of fatty acid mixtures. In fact, the need to determine the composition of mixed fatty acids extracted from plant tissue, to help elucidate their synthetic pathways, was the actual incentive that provoked the development of the technique in the first place. In the first instrument, the column eluent was bubbled through a suitable aqueous liquid to absorb the eluted acids. The solution contained an indicator, the color of which changed as each solute was eluted, and the solution was then manually titrated. Later the titration process was automated by the inventors (probably the first automatic titration apparatus to be made and certainly the only one available at that time), and an integral chromatogram was formed by plotting the volume of base solution added against time. The resulting integram displayed each substance as a step, the height of which was proportional to the amount of fatty acid eluted. Subsequently, Martin developed the density balance detector [2], the first inline detector, which had a very useful linear response. The gas density balance was an extremely complicated and ingenious device consisting of a Wheatstone network of capillary tubes that were drilled out of a high conductivity copper block. Consequently, the sensing device was fairly compact. In the block, there were two columns of gas, one containing the column eluent, the other a reference gas. When solute vapor was present in the column eluent, the pressure difference across the two columns, due to the differing densities of the gases, was arranged to cause a flow of gas over two heated thermocouples, cooling one and heating the other. The output from the thermocouples was fed to an appropriate recording milliammeter. The detector was linear over about three orders of magnitude of concentration and had a sensitivity (minimum detectable concentration) of about 5 X 10– 7 g/ml (n-heptane). The detector output provided a differential output that displayed solute peaks in the conventional manner and could be assessed quantitatively using peak areas or peak heights (methods will be discussed later).

In the early days of gas chromatography little attention was paid to sample preparation, precision sampling, standard selection, etc. Chemists were so elated to be able, quite unexpectedly, to obtain apparently miraculous separations of hitherto completely unresolvable mixtures in a relatively short period of time that the accuracy and precision of the quantitative measurements initially became of somewhat secondary importance. Sensitivity, however, was of great interest and a number of new detectors were rapidly developed. The next GC detector to be developed was the katharometer detector [3] in 1954 (now also known as the thermal conductivity detector and the hot wire detector). This was another, relatively low sensitivity detector (about the same as the density balance) and was quickly followed by another detector of similar sensitivity, the flame thermocouple detector, developed by Scott in 1956 [4]. The last of the early detectors of limited sensitivity was the first of the ionization detectors, the cross-section detector, described by Boer also in 1956 [5]. Subsequent to 1956, the age of the high sensitivity GC detectors began and the first to make its appearance was the ubiquitous flame ionization detector (FID) described by McWilliams [6] in 1958. This was to become the workhorse of all GC analyses, having an extremely high sensitivity and a linear dynamic range exceeding five orders of magnitude. The FID was followed by the relatively specific flame luminosity detector by Grant [7] and finally the exciting family of ionization detectors summarized by Lovelock [8] in 1960. These detectors comprised the macro-argon detector, micro-argon detector and probably the most sensitive detector available, beitmay a specific detector, the electron capture detector. Correctly designed and operated the argon detectors can have sensitivities at least one order of magnitude greater than the FID and the electron capture detector nearly two orders of magnitude greater than the FID.

Subsequent to the burst of innovation that provided most of the detectors in common use today for quantitative gas chromatography, attention was at last turned to the other parts of the gas chromatograph that had an impact on quantitative accuracy. Although some other detectors were developed (e.g., the nitrogen phosphorus detector, a modification of the FID), consideration was now given to the design of accurate and reproducible sampling systems, columns which were appropriately inert to the samples that they were to separate and finally to the methods required for processing the data provided by the detector

The development of quantitative liquid chromatography (LC) has been much slower and initially lagged behind GC by almost a decade. Quantitative LC had to await the development of first, sensitive detectors, second, columns with adequate efficiency, third, high pressure sampling systems that could be used with such columns and fourth, mobile phase supply systems that could provide accurate solvent flow rates at the necessary high pressures, These were all serious engineering challenges, the development of suitable detectors being the least of the problems, High pressure sampling was solved by the introduction of the internal and external loop valves. These were rotary valves consisting of two discs with finely machined contact faces held together by very strong springs. This permitted the valve to be rotated to allow the different ports to be matched without leaking. Columns with the necessary high efficiencies were eventually produced by packing tubes with very small particles (initially 10 µm in diameter) and operating them at very high pressures. The construction of such columns also demanded the development of slurry packing methods to achieve stable beds. High pressure pumps were also required that could be manufactured at a reasonable cost. As a result, the single stroke reciprocating piston pump was developed, which was made from a stainless steel cylinder and a sapphire piston fitted with non-return valves consisting of sapphire balls and seats. Eventually, dual pumps were produced with specially devised driving cams to reduce pump pulsation,

The LC detectors that were developed were many orders of magnitude less sensitive than their GC counterparts and had significantly smaller linear dynamic ranges. The early detectors were, nevertheless, sufficiently sensitive and linear to allow accurate quantitative analysis to be carried out, and also to aid in the development of better LC columns. It is interesting to note that the first LC detector to be developed was by the inventor of the first GC detectors, A.J.P. Martin but in this case with his coworker S.S.Randall [9]. Martin’s device was an electrical conductivity detector which was used with the old type Tswett columns.

The first practical detector that could be used for quantitative work was the refractive index detector developed by Tiselius and Claesson [10] that monitored the change in the refractive index of the column eluent when a solute was present. The design of this detector was extended by Zaukelies and Frost [11] and Vandenheuval and Sipas [12]. Modifications based on the work of Christiansen [13] and the interference detector first described by Bakken and Stenberg [14] were also developed into refractive index-based detectors. Today, despite the many forms of the refractive index detector, including the thermal lens detector of Gorden et al. [15], the most common design used for quantitative analysis is very similar to that originally devised by Vandenheuval and Sipas. The refractive index detector has limited sensitivity and a restricted linear dynamic range but still survives midst the modern technology of LC detectors, largely on account of its catholic response. It is still used, on occasion, to detect the many substances to which other detectors do not respond.

The detector that is most often used in quantitative LC is the UV detector similar to the basic design originally described by Horvath and Lipsky [16]. UV absorption detectors respond to those substances that absorb light in the range 180 to 350 nm. A great number of substances absorb light in this wavelength range, including those substances having one or more double bonds (π electrons) and substances having unshared (unbonded) electrons, e.g. all olefins, all aromatics and compounds, for example, containing > C=O, > C=S, –N=N– groups. The UV light passes through a cell carrying the column eluent and falls on a photo-electric cell (or array) the output of which is conveyed to an appropriate signal modifying amplifier and thence to a recorder or data acquisition system. There are a number of different types of UV detector the details of which will be discussed later. This detector, like the FID in gas chromatography, is the workhorse of quantitative LC analysis. It has reasonable sensitivity (many orders less than the FID) and a linear dynamic range of about three orders of magnitude.

The next detector to be developed was the fluorescent detector, which has a relatively high sensitivity (compared with the UV detector but not the FID). When light is absorbed by a molecule, a transition to a higher electronic state takes place and this absorption is highly specific for the molecules concerned If the excess energy is not dissipated rapidly by collision with other molecules or by other means, the electron will return to the ground state with the emission of light at a lower frequency and the substance is said to fluoresce. As some energy is always lost before emission occurs, the wavelength of the fluorescent light is always greater than the incident light [17]. The popularity of this detector resides largely in its specificity and consequently high sensitivity, which, to some extent, arises from the very low background signal (noise level) that is inherent with this type of detector. Unfortunately its linea...

Table of contents

• Cover Page
• Title Page
• Copyright Page
• Preface
• Part 1: Introduction to Quantitative Chromatographic Analysis
• Part 2: Quantitative Gas Chromatographic Analysis
• Part 3: Quantitative Liquid Chromatography Analysis
• Part 4: Thin Layer Chromatography