1、DEUTSCHE NORM51425Translation by DIN-Sprachendienst.In case of doubt, the German-language original should be consulted as the authoritative text. No part of this translation may be reproduced without the prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Ber
2、lin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).January 2004General principles governing high-performance liquid chromatographic analysis of petroleum products and related substancesDocument comprises 11 pages.ICS 75.160.20Prfung von Minerall-Kohlenwasserstoffen und ve
3、rwandten Stoffen Hochdruckssig-chromatographische Analyse Allgemeine ArbeitsgrundlagenEnglish price group 10 www.din.de www.beuth.de06.05 9632089!,b7|“In keeping with current practice in standards published by the International Organization for Standardization (ISO), a comma has been used throughout
4、 as the decimal marker.ForewordThis standard has been prepared by Technical Committee Prfung von ssigen Kraftstoffen und Heizlen of the Fachausschuss Minerall- und Brennstoffnormung of the Normenausschuss Materialprfung (Mate-rials Testing Standards Committee).1 ScopeThe method specied in this stand
5、ard serves to analyse petroleum products and related organic sub-stances, particularly the soluble sample components in the eluent, by high-performance liquid chromatog-raphy (HPLC). The standard also denes a number of relevant concepts.High-performance liquid chromatography provides information abo
6、ut the components of a sample and their concentration, thus making it possible to determine its composition and to separate substance groups for further analytical examinations.Other types of HPLC, such as ion chromatography and supercritical uid chromatography (SCF) are covered elsewhere.2 Normativ
7、e referencesThis standard incorporates, by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate places in the text, and the titles of the publications are listed below. For dated references, subsequent amendments to or revisions of a
8、ny of these publications apply to this standard only when incorporated in it by amendment or revision. For undated references, the latest edition of the publication referred to applies.DIN 1310 Concepts and symbols relating to the composition of gaseous, liquid and solid mix-turesDIN 32645 Establish
9、ing the limit of detection and quantication under repeatability conditions in chemical analysis Terminology, methods and evaluation.DIN EN ISO 4259 Petroleum products Determination and application of precision data in relation to methods of test (ISO 4259 : 1992 + Corr 1 : 1993)ASTM E 355-96 Standar
10、d practice for gas chromatography terms and relationships 1)1 Rohrschneider, L. Grundlagen chromatographischer Trennverfahren (Principles of chromatographic separation methods), Ullmann, 1980: Verlag Chemie, vol. 5, 105.2 Nomenclature for Chromatography (IUPAC Recommendation 1993).1) Obtainable from
11、 Beuth Verlag GmbH, 10772 Berlin, Germany.Page 2 DIN 51425 : 2004-013 Principles3.1 High-performance liquid chromatography3.1.1 GeneralThe term high-performance liquid chromatography (HPLC) refers to chromatographic methods in which the mobile phase (eluent) is liquid and the stationary phase is con
12、tained in the column (see subclause 3.1.3).NOTE: The modern liquid chromatographic procedure is referred to as being high-performance because it achieves a high efciency using very small particles and a fairly high inlet pressure.The stationary phase may be either a solid phase or a gel, the former
13、being composed predominantly of spheri-cal granular material normally having a particle size of 3 m to 10 m. A distinction is made between modied and unmodied separating phases. The latter are normally composed of silica gel or aluminium oxide (sorbent), while in the case of the former the separatin
14、g layer is chemically bound to a support material, an example being a silica gel modied with alkyl groups.The mobile phase passes through the stationary phase in one direction. The components of the sample to be analysed must be completely soluble in the eluent, the latter being selected primarily f
15、or optimum elution efciency and good selectivity.After the sample has been injected at the column inlet, its components are swept through or eluted from the column by the mobile phase. The sample components are analysed by a detector at the outlet of the column.3.1.2 Adsorption chromatographyIn adso
16、rption chromatography, separation is based on differences in the distribution of the analyte between mobile phase and sorbent.3.1.3 Partition chromatographyIn partition chromatography, separation is based on differences in solubility in the mobile phase and stationary phase (modied separating phase)
17、.3.1.4 Size exclusion chromatographySize exclusion chromatography (SEC) is based on the partial exclusion of the analyte from the interior of the porous support material owing to differences in size, shape or charge.NOTE: When using swollen gels, this technique is also referred to as gel ltration ch
18、romatography (GFC) or gel permeation chromatography (GPC).3.1.5 Normal phase chromatographyIn normal phase chromatography (NPC), the stationary phase (e.g. silica gel or aluminium oxide) is more polar than the eluent.3.1.6 Reversed phase chromatographyIn reversed phase chromatography (RPC), the mobi
19、le phase is substantially more polar than the stationary phase (e.g. silica gel modied with alkyl groups).3.2 High-performance liquid chromatogram3.2.1 GeneralA high-performance liquid chromatogram is a plot of the detector signal against time. The concepts given in subclauses 3.2.2 to 3.2.7 are use
20、d when referring to high-performance liquid chromatograms (see gure 1).Page 3 DIN 51425 : 2004-01Key:A Peak areaABPeak area of component Bb0,5Peak width at half heighth Peak heighttMDead timetR,ARetention time of component AtR,BRetention time of component BtqR,ARetention time of component A adjusted
21、 for the dead timetqR,BRetention time of component B adjusted for the dead timew Peak widthFigure 1: High-performance liquid chromatogram3.2.2 Zero lineThe line recorded by the plotter or computer during the ow of pure eluent.3.2.3 BaselineAn imaginary line underneath a peak or a group of superimpos
22、ed peaks obtained by connecting the consecutive minima of a gas chromatographic curve. It is generally assumed to be a straight line, but it does not necessarily coincide with the zero line. *)3.2.4 PeakThe portion of a chromatogram that deviates from the baseline when a single component is eluted f
23、rom the column, as recorded by a plotter or computer. If there are a number of components present in a sample, peaks may be superimposed.3.2.5 Peak heightThe distance between the peak maximum and the baseline (designated by h).3.2.6 Peak width at half heightThe width of a peak measured at half its h
24、eight (designated by b0,5).3.2.7 Peak areaThe area, A, enclosed between a peak and the baseline (e.g. ABin gure 1). If peaks are superimposed, par-ticular procedures may need to be specied for determining the peak areas when using the individual test methods, a frequently used procedure being to det
25、ermine the peak areas by dropping the perpendiculars at the minima.Depending on the type of detector used, the unit to measure the peak area will be ampere-second or volt-second.*) Translators note. In ASTM E 355 dened as the portion of a chromatogram recording the detector response in the absence o
26、f solvent eluted from the column.Page 4 DIN 51425 : 2004-013.3 Retention parameters3.3.1 Retention timeThe retention time, tR, of the component to be determined is the time between the injection of the sample and the detection of the peak maximum for the component. The retention time can be resolved
27、 into the dead time, tM, (see subclause 3.3.3) necessary for the elution of an unretarded component that does not interact with the stationary phase, and the adjusted retention time, tqR, which is the retention time less the dead time (see subclause 3.3.2.1) and is a measure of the extent to which t
28、he sample components interact with the stationary phase.3.3.2 Retention ratioThe retention ratio, ri, is the ratio of the adjusted retention time of substance i, tqR,i, to the adjusted retention time of a standard (St), tqR,St, i.e.:rttiR,iR,St=(1)NOTE: For practical reasons, the standard should be
29、readily obtainable and elute in the vicinity of the sub-stance to be determined.The retention ratio which relates to a particular stationary phase, a particular eluent composition and a particu-lar temperature, is largely independent of equipment parameters under isothermal and isocratic conditions
30、and is consequently suitable for characterizing the separation characteristics of this phase.3.3.3 Retention volumeThe volume of mobile phase, VR, eluted in the time between the injection of the sample and the detection of a peak maximum, tR, while the dead volume, VM, is the volume eluted at the de
31、ad time.NOTE: The dead volume can be used to calculate the ow rate of the eluent in the column 2.3.3.4 Linear velocity of eluentRatio, u, of the length of the column, L, to the dead time, i.e.:uLt=M(2)3.4 Peak resolutionThe resolution of two adjacent peaks, R, is dened as:Rt tb b=+1180 5 0 5,( ) ( )
32、R,B R,A, A , B(3)NOTE 1: In this equation, R is dened as the ratio of the peak separation, tR,B tR,A, to the mean peak width, (wA+ wB)/2. For Gaussian peaks, w is equal to 1,699 b0,51.NOTE 2: Taking account of the fact that the resolution of two adjacent peaks is also affected by the length and inte
33、rnal diameter of the connecting capillaries downstream of the injector, the use of short capillaries of small internal diameter (e.g. 0,12 mm) is recommended.3.5 Plate numberThe plate number (formerly theoretical plate number), N, is a measure of column efciency and is given by:Ntb=5 5450 52,R,(4)Th
34、e effective plate number, Neff, is calculated using the adjusted retention time instead of the total retention time, and is given by:NtbeffR,=5 5450 52(5)NOTE: These parameters are dependent on the eluent and the sample components.Page 5 DIN 51425 : 2004-013.6 Peak skewPeak skew, PS, is a criterion
35、describing the quality of separation by HPLC and is shown in gure 2. It is given by:PSME MBBE=(6)whereME is the distance between points M and E;MB is the distance between points M and B;BE is the distance between points B and E.If PS is less than zero (negative), the peak is fronted (see gure 2), if
36、 it is greater than zero, the peak is tailed, and if it equals zero, the peak is symmetrical.Since peak skew is always referred to in connection with particular separation problems, the above denition gives an adequate description of a requirement for a separation system on a case-by-case basis.Figu
37、re 2: Peak skewNOTE: Asymmetrical peaks occur if there is no linear relationship between the distribution of the sample com-ponents between the mobile and stationary phases, and the concentration. This may be due, for example, to the column being overloaded with an excessively large sample quantity,
38、 or to the stationary phase or eluent, or to the temperature in the column being inappropriate, and to an insufcient deactivation of the separation system. In such cases, quantity-dependent changes may occur in the retention parameters, the plate number and the peak resolution.3.7 UnitsAs specied in
39、 DIN 1310, all results and calibration factors shall be reported in SI units or combinations of such units (e.g. g/100 g, g/g or mg/kg for proportions by mass, v, ml/100 ml or ml/m3for proportions by volume, f, and mol/mol for mole fractions, x).4 Apparatus4.1 High-performance liquid chromatograph4.
40、1.1 GeneralThe test equipment used shall be a commercial high-performance liquid chromatograph consisting of the com-ponents described below (see gure 3).Page 6 DIN 51425 : 2004-01Key:1 Eluent reservoir 18 Sample injection system2 Sintered metal frit 19 Precolumn3 High pressure pump 10 Separating co
41、lumn4 Pulse damper 11 Temperature-controlled oven5 Flushing valve 12 Detector6 Manometer 13 Recorder and evaluation system7 Scavenger column 14 Fraction collectorFigure 3: Example of an isocratic high-performance liquid chromatographOther high-performance liquid chromatographs may be used if they co
42、nform to this standard or meet the requirements specied in the individual test standards.4.1.2 Pump systemsThe high-performance liquid chromatograph shall be equipped with a pump system capable of establishing a reproducible eluent ow rate.4.1.2.1 EluentThe eluents required are usually supplied from
43、 reservoirs.Since impurities in the eluents may interact with the stationary phase, the type of eluent and the purity require-ments relating to it shall suit the particular task and be specied in the relevant test standards.NOTE: It may be necessary to degas the eluent (e.g. using helium or vacuum),
44、 or to dry it (e.g. by adding suit-able molecular sieves).4.1.2.2 Eluent mixingIsocratic or gradient elution may be used. In the former, the composition of the mobile phase remains constant throughout the entire elution, while, in the latter, the composition of the mobile phase is varied continuousl
45、y or in steps (stepwise gradient).4.1.3 Sample inlet systems4.1.3.1 GeneralSample inlet systems are intended to introduce liquid samples into the eluent ow in a reproducible manner. The sample should be applied to the top of the column as a narrow band. Sample loops are especially suitable for sampl
46、e injection. Details are to be specied in the relevant test standards.4.1.3.2 Automatic injection systemsAutomatic injection systems may be used if they meet the requirements specied in the relevant test standards. Due attention shall be paid to the manufacturers instructions.Page 7 DIN 51425 : 2004
47、-014.1.4 Column4.1.4.1 GeneralThe column is a metal tube containing the stationary phase for separating the sample components. Analyti-cal columns normally have an internal diameter of 2 mm (narrow bore) to 4,6 mm, while SEC and preparative columns may have a greater internal diameter.4.1.4.2 Precol
48、umnPrecolumns are used to keep non-elutable components out of the separation column and shall be changed at regular intervals or whenever necessary.4.1.4.3 Scavenger columnThe scavenger column is intended to saturate the eluent with stationary phase.4.1.4.4 Column switching techniquesColumn switchin
49、g techniques are used, for example,a) to increase the separation efciency of the system using additional columns to separate unseparated peaks (multidimensional chromatography),b) to shorten the analysis time by back-ushing components that are difcult to elute and determining them jointly in a detector.Suitable devices for switching the columns are valves that have a small switching volume compared with the peak volume and do not interact with the sample.4.1.5 Temperature control unitsTemperature c
