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Study Notes: Sample Preparation Background for Chromatographic and Electrophoretic Analysis

Sample preparation includes all of those procedures the analyst performs on the sample to make it ready for the actual ‘measurement’ stage of analysis.

We shall now explore general aspects of sample preparation procedures used in chromatographic and electrophoretic analysis. In essence, the aim is to convert the sample into a form that is suitable to be measured by the instrument. This aspect is as important as knowing how to operate the instrument. That test samples are representative and homogeneous, is assumed in this discussion.

A photograph of a gas chromatograph instrument commonly found in an analytical laboratory. Photograph that shows some detail of the HPLC equipment.

In considering sample preparation there are a number of general factors that need to be addressed.

  • Is the sample in solid, liquid or gaseous form?
  • Does the sample contain compounds, including particulate matter, that would interfere with the reading given by the instrument?
  • Is the sample too concentrated?
  • Is the sample too diluted?

There are various considerations that impact these factors, as follows.

  • Analyte properties, organic or inorganic
  • Required detection limit
  • Sample matrix
  • The particular requirements for the instrument.
The primary criterion for chromatographic and electrophoretic techniques is that the sample be presented to the instrument as a liquid, often an aqueous solution. The GC requires a gaseous sample on the column. Either a gaseous sample is injected or a liquid sample is injected which is then vapourised in a special flash vapouriser port. A sample that is liquid in itself may be suitable for measurement ‘as is’ but further pre-treatment may be involved, even if it is only dilution into the measurement range of the instrument. A sample of a solid material as it normally appears and then after making up in solution in a volumetric flask.

True gaseous samples (for instance headspace analysis) will not be considered here.

Solids need to be converted into a soluble form.

  • All that may be necessary is to dissolve the sample in water
  • Where the sample is insoluble an alternative solvent might be sought, such as hexane, to dissolve non-polar organic compounds
  • A solid insoluble sample can be chemically broken down or dissolved in order to release an analyte into solution.

Once in solution, further processing of the sample may still be required.

‘Matrix’ refers to the overall chemical composition as well as the physical properties of the sample in which the analyte resides.

Urine, for example, is a straw coloured liquid consisting predominantly of water that includes a complex mixture of salts, carbohydrates, proteins, some cells and breakdown products of the body’s metabolism.

Substances may be present in the sample, that interfere with the analytical process - called ‘interferences’. Interferences may be chemically masked, or in other words ‘blocked’, to negate their impact or the analyte may need to be separated from its matrix - such processes are known as ‘sample clean up’.

In some cases, especially for the use of standards and control substances, the standard or control may need to be dissolved in a matrix as near to identical as the matrix in which the substance is received.

For example, a standard for the metabolic by-product of a heart drug that is detected in urine will need to be dissolved in a representative sample of urine before analysis. This ensures any discrepancies are not due to the lack of a ‘matrix effect’ in the standard. You may well be asked to donate blood or urine for matrix pools if you work in a pathology laboratory. Please donate with good grace for the common good of the laboratory!

Detection limit (or sensitivity) concerns the concentration level down to which an analysis must be able to measure for the purpose of the analysis. A detection limit could be 0.1 µg/L or only as low as 1 mg/L. An equation showing that point one micro gram per litre is ten thousand times smaller than one milligram per litre.

Improvements to analytical methods that result in lower detection limits and shorter analysis times often arise from improvements to sample preparation. To illustrate this, imagine a biological toxin that has been discovered and is active at concentrations lower than normal. A new approach to sample preparation might be developed to include steps that chemically alter the toxin by attachment of an organic reagent or an antibody directed against the toxin (an antitoxin). The choice of an appropriate reagent can significantly enhance sensitivity (ie lower the detection limit) and selectivity (ie reduce the impact on the analyte from interfering compounds). ‘Chemical derivatisation’ is the name of this type of procedure when a chemical reagent is employed.

Sample preparation can be tedious and time consuming. Using acid to break down (digest) a sample of a solid insoluble material could take some hours compared to seconds to acquire the output from an instrument.

The more steps involved in sample preparation, the greater the potential for loss of sample with consequent negative effects on accuracy and precision. For quantitative analysis, the number of steps needs to be optimal and the analyst needs to perform the work with due care.

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