ASTM E1303-95(2005)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation:E1303–95(Reapproved2005)
Standard 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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope circumstances it might also be necessary to test the detector in
the separation mode with an LC column in the system, and the
1.1 This practice covers tests used to evaluate the perfor-
appropriate concerns are also mentioned. The terms and tests
mance and to list certain descriptive specifications of a
described in this practice are sufficiently general so that they
refractive index (RI) detector used as the detection component
may be adapted for use at whatever conditions may be chosen
of a liquid chromatographic (LC) system.
for other reasons.
1.2 This practice is intended to describe the performance of
the detector both independent of the chromatographic system
4. Noise, Drift, and Flow Sensitivity
(static conditions, without flowing solvent) and with flowing
4.1 Descriptions of Terms Specific to This Standard:
solvent (dynamic conditions).
4.1.1 short term noise—this noise is the mean amplitude in
1.3 The values stated in SI units are to be regarded as the
refractive index units (RIU) for random variations of the
standard.
detector signal having a frequency of one or more cycles per
1.4 This standard does not purport to address all of the
minute. Short term noise limits the smallest signal detectable
safety concerns, if any, associated with its use. It is the
by an RI detector, limits the precision attainable, and sets the
responsibility of the user of this standard to establish appro-
lower limit on the dynamic range. This noise corresponds to
priate safety and health practices and determine the applica-
observednoiseoftheRIdetectoronly.(Theactualnoiseofthe
bility of regulatory limitations prior to use.
LC system may be larger or smaller than the observed value,
2. Referenced Documents depending upon the method of data collection, or signal
2 monitoringofthedetector,sinceobservednoiseisafunctionof
2.1 ASTM Standards:
the frequency, speed of response and the band width of the
E386 Practice for Data Presentation Relating to High-
recorder or other electronic circuit measuring the detector
ResolutionNuclearMagneticResonance(NMR)Spectros-
signal.)
copy
4.1.2 long term noise—this noise is the maximum ampli-
3. Significance and Use
tude in RIU for random variations of the detector signal with
frequencies between 6 and 60 cycles per h (0.1 and 1.0 cycles
3.1 Although it is possible to observe and measure each of
per min). It represents noise that may be mistaken for a
several characteristics of a detector under different and unique
late-eluting peak.This noise corresponds to the observed noise
conditions,itistheintentofthispracticethatacompletesetof
only and may not always be present.
detector test results should be obtained under the same oper-
4.1.3 drift—the average slope of the long term noise enve-
ating conditions. It should also be noted that to specify
lope expressed in RIU per hour as measured over a period of
completely a detector’s capability, its performance should be
1h.
measured at several sets of conditions within the useful range
4.1.4 static—refers to the noise and drift measured under
of the detector.
conditions of no flow.
3.2 Theobjectiveofthispracticeistotestthedetectorunder
4.1.5 dynamic—refers to the noise and drift measured at a
specified conditions and in a configuration without an LC
flow rate of 1.0 mL/min.
column. This is a separation independent test. In certain
4.1.6 flow sensitivity—the rate of change of signal displace-
ment (in RIU) vs flow rate (in mL/min) resulting from step
This practice is under the jurisdiction ofASTM Committee E13 on Molecular
changes in flow rate calculated at 1 mL/min as described in
Spectroscopy and Chromatography and is the direct responsibility of Subcommittee
4.3.12.
E13.19 on Chromatography.
4.2 Test Conditions:
Current edition approved Feb. 1, 2005. Published March 2005. Originally
approved in 1989. Last previous edition approved in 2000 as E1303–95(2000).
4.2.1 Thesametestsolventmustbeusedinbothsampleand
DOI: 10.1520/E1303-95R05.
reference cells. The test solvent used and its purity should be
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
specified. Water equilibrated with the laboratory atmosphere
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E1303–95 (2005)
containingminimumimpuritiesisthepreferredtestsolventfor besetsuchthattheamplitudeofshorttermnoisemaybeeasily
measuring noise and drift. Water for this purpose (preferably measured. Ideally, the output should contain no filtering of the
purified by distillation, deionization, or reverse osmosis) signal. If the filtering cannot be turned off, the minimum time
should be drawn, filtered through a 0.45-µm filter, and allowed constant should be set and noted in the evaluation. Manuals or
to stand in a loosely covered container for several hours at manufacturers should be consulted to determine if time con-
ambient temperature in the laboratory in which testing is to be stant and detector range controls are coupled, and information
carried out. This will ensure complete equilibration of the should be obtained to determine if they can be decoupled for
water with 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 3
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.
the sample and the trapped reference can lead to significant errors in
4.3.5 Draw pairs of parallel lines, each between ⁄2 to 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 (5.2.9). 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.
Theovenshouldbesetatasuitabletemperature,followingthe
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.
4.2.3 Linearity and speed of response of the recorder or
other data acquisition device used should be such that it does
not distort or otherwise interfere with the performance of the
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;ifsignificantlywider,itshouldbereplacedforthistest.
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
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
E1303–95 (2005)
measurement. Draw a series of parallel lines to these centers,
each 10 min in length (Fig. 1), and choose that pair of lines
whose distance apart perpendicular to the time axis is greatest.
This distance is the static long term noise.
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/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
FIG. 3 Example of Plot for Calculation of Flow Sensitivity
noise following the procedure outlined in 4.3.6.
4.3.11 Draw the pair of parallel lines in accordance with
4.3.7. The slope of this line is the dynamic drift.
sensitivityinRIUmin/mL.Itispreferredtogivethenumerical
4.3.12 Stopthechromatographicflow.Allowatleast15min
value and show the plot as well.
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/min. Run for 15 min, or more if necessary for
re-equilibration,ataslowrecorderspeed.Increasetheflowrate 5.1 Descriptions of Terms Specific to this Standard:
to 1.0 mL/min and record for 15 min or more. Run at 2.0, 4.0, 5.1.1 minimum detectability—that concentration of a spe-
and 8.0 mL/min if the pressure flow limit of the chromato- cific solute in a specific solvent that gives a signal equal to
graphic system is not exceeded.Ifnecessary,adjustthedetector twice the static short-term noise.
range to maintain an on-scale response. 5.1.1.1 Discussion—The static short-term noise is a mea-
4.3.13 Draw a horizontal line through the plateau produced surement of peak-to-peak noise.Astatistical approach to noise
at each flow rate, after a steady state is reached (Fig. 2). suggeststhatavalueofthreetimestherms(root-mean-square)
Measure the vertical displacement between these lines, and
noisewouldensurethatanyvalueoutsidethisrangewouldnot
expressinRIU(5.2.9).Plotthesevaluesversusflowrate.Draw be noise with a confidence level of greater than 99%. Since
a smooth curve connecting the points and draw a tangent at 1
peak-to-peak noise is approximately five times the rms
4,5
mL/min (Fig. 3). Express the slope of the line as the flow noise, the minimum detectability defined in this practice is a
more conservative estimate. Minimum detectability, as defined
in this practice, should not be confused with the limit of
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
Blair, E. J., Introduction to Chemical Instrumentation, McGraw-Hill, New
FIG. 2 Example for the Measurement of Flow Sensitivity York, NY, 1962, and Practice E386.
E1303–95 (2005)
linearity plot specified in 5.2.13. The linear range may be solution is 50 times the concentration of the normal solution
expressed in three different ways: (5.2.2) used for calibration and is assigned a normalized
5.1.3.1 As the ratio of the upper limit of linearity obtained concentration of 50.
from the linearity plot, and the minimum linear concentration, 5.2.4 Serially dilute the stock solution (5.2.2) to 0.01
both measured for the same test substance in the same test relative concentration according to Table 1. Use the stock
solvent as follows: solution and the diluted solutions for linearity and dynamic
range testing.
L.R. 5 C /C (2)
max min
5.2.5 Because atmosphere-equilibrated water is used as the
where:
mobile phase and sample diluent for this procedure, it is
L.R. = linear range of the detector,
advisable to apply a slight back pressure to the sample cell to
C = upper limit of linearity obtained from th
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