ASTM E1303-95(2000)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation:E1303–95 (Reapproved 2000)
Practice for
Refractive Index Detectors Used in Liquid Chromatography
This standard is issued under the fixed designation E1303; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope described in this practice are sufficiently general so that they
may be adapted for use at whatever conditions may be chosen
1.1 This practice is intended to describe tests used to
for other reasons.
evaluate the performance and to list certain descriptive speci-
fications of a refractive index (RI) detector used as the
4. Noise, Drift, and Flow Sensitivity
detectioncomponentofaliquidchromatographic(LC)system.
4.1 Descriptions of Terms Specific to This Standard:
1.2 This practice is intended to describe the performance of
4.1.1 short term noise—Thisnoiseisthemeanamplitudein
the detector both independent of the chromatographic system
refractive index units (RIU) for random variations of the
(static conditions, without flowing solvent) and with flowing
detector signal having a frequency of one or more cycles per
solvent (dynamic conditions).
min. Short term noise limits the smallest signal detectable by
1.3 The values stated in SI units are to be regarded as the
anRIdetector,limitstheprecisionattainableandsetsthelower
standard.
limitonthedynamicrange.Thisnoisecorrespondstoobserved
1.4 This standard does not purport to address all of the
noise of the RI detector only. (The actual noise of the LC
safety concerns, if any, associated with its use. It is the
system may be larger or smaller than the observed value,
responsibility of the user of this standard to establish appro-
depending upon the method of data collection, or signal
priate safety and health practices and determine the applica-
monitoringofthedetector,sinceobservednoiseisafunctionof
bility of regulatory limitations prior to use.
the frequency, speed of response and the band width of the
2. Referenced Documents recorder or other electronic circuit measuring the detector
signal.)
2.1 ASTM Standards:
4.1.2 long term noise—this noise is the maximum ampli-
E386 Practice for Data Presentation Relating to High-
tude in RIU for random variations of the detector signal with
ResolutionNuclearMagneticResonance(NMR)Spectros-
frequencies between 6 and 60 cycles per h (0.1 and 1.0 cycles
copy
per min). It represents noise that may be mistaken for a
3. Significance and Use late-eluting peak.This noise corresponds to the observed noise
only and may not always be present.
3.1 Although it is possible to observe and measure each of
4.1.3 drift—the average slope of the long term noise enve-
several characteristics of a detector under different and unique
lope expressed in RIU per hour as measured over a period of
conditions,itistheintentofthispracticethatacompletesetof
1h.
detector test results should be obtained under the same oper-
4.1.4 static—refers to the noise and drift measured under
ating conditions. It should also be noted that to specify
conditions of no flow.
completely a detector’s capability, its performance should be
4.1.5 dynamic—refers to the noise and drift measured at a
measured at several sets of conditions within the useful range
flow rate of 1.0 mL/min.
of the detector.
4.1.6 flow sensitivity—the rate of change of signal displace-
3.2 Theobjectiveofthispracticeistotestthedetectorunder
ment (in RIU) vs flow rate (in mLper min) resulting from step
specified conditions and in a configuration without an LC
changesinflowratecalculatedat1mLperminasdescribedin
column. This is a separation independent test. In certain
4.3.12.
circumstances it might also be necessary to test the detector in
4.2 Test Conditions:
the separation mode with an LC column in the system, and the
4.2.1 Thesametestsolventmustbeusedinbothsampleand
appropriate concerns are also mentioned. The terms and tests
reference cells. The test solvent used and its purity should be
specified. Water equilibrated with the laboratory atmosphere
This practice is under the jurisdiction ofASTM Committee E13 on Molecular
containingminimumimpuritiesisthepreferredtestsolventfor
Spectroscopy and is the direct responsibility of Subcommittee E13.19 on Chroma-
measuring noise and drift. Water for this purpose (preferably
tography.
purifiedbydistillation,deionizationorreverseosmosis)should
Current edition approved May 15, 1995. Published July 1995. Originally
published as E1303–89. Last previous edition E1303–89.
Annual Book of ASTM Standards, Vol 03.06.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E1303
bedrawn,filteredthrougha0.45µmfilterandallowedtostand constant should be set and noted in the evaluation. Manuals or
in a loosely covered container for several hours at ambient manufacturers should be consulted to determine if time con-
temperature in the laboratory in which testing is to be carried stant and detector range controls are coupled, and information
out. This will ensure complete equilibration of the water with should be obtained to determine if they can be decoupled for
the gases in the laboratory atmosphere. testing. Set the recorder zero to near mid-scale. Record at least
1hofbaselineunderthesestaticconditions,duringwhichtime
NOTE 1—It is essentially impossible to maintain a constant RI value of
the ambient temperature should not change by more than 2°C.
de-gassedwaterandofverydilutesamplesinde-gassedwater.Thisisdue
to the fact that the difference in refractive index between completely
NOTE 2—RI detectors will have one or more controls labeled attenua-
−6
de-gassed water and atmosphere-equilibrated water is 1.5 310 RIU.
tion, range, sensitivity, and scale factor. All are used to set the full scale
Thus, small differences in the concentration of dissolved gases between
range (in RIU) of an output display device such as a strip chart recorder.
sample and the trapped reference can lead to significant errors in
4.3.5 Draw pairs of parallel lines, each between ⁄2to 1 min
measurement of solutions where the expected difference in RI due to
−6
in length, to form an envelope of all observed random
solute is of the order of 10 RIU or less. Therefore, in order to minimize
error in determining samples with small RIU differences between them,
variations over any 15 min period (Fig. 1). Draw the parallel
atmosphere-equilibrated water (5.2.1) is recommended as the solvent for
lines in such a way as to minimize the distance between them.
determining linearity and minimum detectability (Section 5).
Measure the distance perpendicular to the time axis between
4.2.2 The detector should be located at the test site and
the parallel lines. Convert this value to RIU (4.2.9). Calculate
switched on at least 24 h prior to the start of testing. Some the mean value over all the segments; this value is the static
detectors provide an oven to thermostat the optics assembly.
short term noise.
The oven should be set at a suitable temperature, following the 4.3.6 Now mark the center (center of gravity) of each
manufacturer’s recommendations, and this temperature should
segment over the 15 min period of the short term noise
be noted and maintained throughout the test procedures. measurement. Draw a series of parallel lines to these centers,
4.2.3 Linearity and speed of response of the recorder or
each 10 min in length (Fig. 1), and choose that pair of lines
other data acquisition device used should be such that it does whose distance apart perpendicular to the time axis is greatest.
not distort or otherwise interfere with the performance of the
This distance is the static long term noise.
detector. Ifadditionalamplifiersareusedbetweenthedetector
and the final readout device, their characteristics should also
first be established.
4.3 Methods of Measurement:
4.3.1 Connecta1m (39.37 in.) length of clean, dry,
stainless steel tubing of 0.25 mm (0.009 to 0.01 in.) inside
diameter in place of the analytical column. The tubing can be
straight, or coiled to minimize the space requirement. The
tubingshouldterminateinstandardlowdeadvolumefittingsto
connect with the detector and to the pump. Commercial
chromatographs may already contain some capillary tubing to
connect the pump to the injection device. If this is of a similar
diameter to that specified, it should be included in the 1.0 m
length; if significantly wider, it should be replaced for this test.
4.3.2 Repeatedly rinse the reservoir and chromatographic
system,includingthedetector,withthetestsolventpreparedas
described in 4.2.1, until all previous solvent is removed from
the system. Fill the reservoir with the test solvent.
4.3.3 Thoroughly flush the reference cell with the same
solvent; keep the reference cell static.
4.3.3.1 It may be necessary to flush both sample and
reference cells with an intermediate solvent (such as methanol
or acetone), if the solvent previously used in the system is
immiscible with the test solvent.
4.3.4 Allow the chromatographic system to stabilize for at
least 60 min without flow. The detector range, Note 2, should
besetsuchthattheamplitudeofshorttermnoisemaybeeasily
measured. Ideally, the output should contain no filtering of the
signal. If the filtering cannot be turned off, the minimum time
Munk, M. N., Liquid Chromatography Detectors, (T. M. Vickrey, Ed.), Marcel
Dekker, New York and Basel, 1983, pp. 165–204.
Bonsall, R. B., “The Chromatography Slave—The Recorder,” Journal of Gas FIG. 1 Examples for the Measurement of Short Term Noise, Long
Chromatography, Vol 2, 1964, pp. 277–284. Term Noise and Drift
E1303
4.3.7 Draw the pair of parallel lines, over the1hof
measurement, that minimizes the distance perpendicular to the
time axis between the parallel lines. The slope of either line,
measured in RIU/h, is the static drift.
4.3.8 Set the solvent delivery system to a flow rate that has
previously been shown to deliver 1.0 mL per min under the
same conditions of capillary tubing, solvent, and temperature.
Allow at least 15 min to stabilize. Set the recorder zero near
mid-scale. Record at least1hof baseline under these flowing
conditions, during which time the ambient temperature should
not change by more than 2°C.
4.3.9 Drawpairsofparallellines,measuretheperpendicular
distances and calculate the dynamic short term noise, in the
manner described in 4.3.5 for the static short term noise.
4.3.10 Make the measurement for the dynamic long term
noise following the procedure outlined in 4.3.6.
4.3.11 Draw the pair of parallel lines as directed in 4.3.7.
The slope of this line is the dynamic drift.
FIG. 3 Example of Plot for Calculation of Flow Sensitivity
4.3.12 Stopthechromatographicflow.Allowatleast15min
for re-equilibration. Set the recorder at about 5% of full scale
and leave the detector range setting at the value used for the
5. Minimum Detectability, Linear Range, Dynamic
noise measurements. Set the solvent delivery system at a flow
Range, and Calibration
rate of 0.5 mL per min. Run for 15 min, or more if necessary
for re-equilibration, at a slow recorder speed. Increase the flow 5.1 Descriptions of Terms Specific to this Standard:
rate to 1.0 mL per min and record for 15 min or more. Run at 5.1.1 minimum detectability—that concentration of a spe-
cific solute in a specific solvent that gives a signal equal to
2.0, 4.0 and 8.0 mL per min if the pressure flow limit of the
twice the static short-term noise.
chromatographic system is not exceeded. If necessary, adjust
5.1.1.1 Discussion—The static short-term noise is a mea-
the detector range to maintain an on-scale response.
surement of peak-to-peak noise.Astatistical approach to noise
4.3.13 Draw a horizontal line through the plateau produced
suggeststhatavalueofthreetimestherms(root-mean-square)
at each flow rate, after a steady state is reached (Fig. 2).
noise would insure that any value outside this range would not
Measure the vertical displacement between these lines, and
be noise with a confidence level of greater than 99%. Since
expressinRIU(4.2.9).Plotthesevaluesversusflowrate.Draw
peak-to-peak noise is approximately five times the rms
a smooth curve connecting the points and draw a tangent at 1
4,5
noise , the minimum detectability defined in this Practice is a
mL per min (Fig. 3). Express the slope of the line as the flow
more conservative estimate. Minimum detectability, as defined
sensitivity in RIU min per mL. It is preferred to give the
in this Practice, should not be confused with the limit of
numerical value and show the plot as well.
detection in an analytical method using a refractive index
detector.
5.1.2 sensitivity (response factor)—the signal output per
unit concentration of the test substance in the test solvent, in
accordance with the following relationship:
S 5 R/C (1)
where:
S = sensitivity (response factor), RIU·L/g,
R = measured detector response, RIU, and
C = concentration of the test substance in the test solvent
g/L.
5.1.3 linear range—the range of concentrations of the test
substance in the test solvent, over which the sensitivity of the
detector is constant to with 5% as determined from the
linearity plot specified in 5.2.13. The linear range may be
expressed in three different ways:
Blair, E. J., Introduction to Chemical Instrumentation, McGraw-Hill, New
FIG. 2 Example for the Measurement of Flow Sensitivity York, NY, 1962, Practice and E386.
E1303
5.1.3.1 As the ratio of the upper limit of linearity obtained solution is 50 times the concentration of the normal solution
from the linearity plot, and the minimum linear concentration, (5.2.2) used for calibration and is assigned a normalized
both measured for the same test substance in the same test concentration of 50.
solvent as follows: 5.2.4 Serially dilute the stock solution (5.2.2) to 0.01
relative concentration according to Table 1. Use the stock
L.R. 5 C /C (2)
max min
solution and the diluted solutions for linearity and dynamic
range testing.
where:
5.2.5 Because atmosphere-equilibrated water is used as the
L.R. = linear range of the detector,
mobile phase and sample diluent for this procedure, it is
C = upper limit of linearity obtained from the linearity
max
advisable to apply a slight back pressure to the sample cell to
plot, g/L, and
prevent outgassing in the cell. This may be safely achieved by
C = minimum linear concentration g/L, as defined in
min
placing the solvent waste container on a shelf above the
5.2.13.1, the minimum linear concentration should
detector. A
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