ASTM E1303-95(2010)
(Practice)Practice for Refractive Index Detectors Used in Liquid Chromatography
Practice for Refractive Index Detectors Used in Liquid Chromatography
SIGNIFICANCE AND USE
Although it is possible to observe and measure each of several characteristics of a detector under different and unique conditions, it is the intent of this practice that a complete set of detector test results should be obtained under the same operating conditions. It should also be noted that to specify completely a detector's capability, its performance should be measured at several sets of conditions within the useful range of the detector.
The objective of this practice is to test the detector under specified conditions and in a configuration without an LC column. This is a separation independent test. In certain circumstances it might also be necessary to test the detector in the separation mode with an LC column in the system, and the appropriate concerns are also mentioned. The terms and tests described in this practice are sufficiently general so that they may be adapted for use at whatever conditions may be chosen for other reasons.
SCOPE
1.1 This practice covers tests used to evaluate the performance and to list certain descriptive specifications of a refractive index (RI) detector used as the detection component of a liquid chromatographic (LC) system.
1.2 This practice is intended to describe the performance of the detector both independent of the chromatographic system (static conditions, without flowing solvent) and with flowing solvent (dynamic conditions).
1.3 The values stated in SI units are to be regarded as the standard.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: E1303 − 95 (Reapproved 2010)
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 3.2 Theobjectiveofthispracticeistotestthedetectorunder
specified conditions and in a configuration without an LC
1.1 This practice covers tests used to evaluate the perfor-
column. This is a separation independent test. In certain
mance and to list certain descriptive specifications of a
circumstances it might also be necessary to test the detector in
refractive index (RI) detector used as the detection component
the separation mode with an LC column in the system, and the
of a liquid chromatographic (LC) system.
appropriate concerns are also mentioned. The terms and tests
1.2 This practice is intended to describe the performance of
described in this practice are sufficiently general so that they
the detector both independent of the chromatographic system
may be adapted for use at whatever conditions may be chosen
(static conditions, without flowing solvent) and with flowing
for other reasons.
solvent (dynamic conditions).
1.3 The values stated in SI units are to be regarded as the 4. Noise, Drift, and Flow Sensitivity
standard.
4.1 Descriptions of Terms Specific to This Standard:
1.4 This standard does not purport to address all of the
4.1.1 short term noise—this noise is the mean amplitude in
safety concerns, if any, associated with its use. It is the
refractive index units (RIU) for random variations of the
responsibility of the user of this standard to establish appro-
detector signal having a frequency of one or more cycles per
priate safety and health practices and determine the applica-
minute. Short term noise limits the smallest signal detectable
bility of regulatory limitations prior to use.
by an RI detector, limits the precision attainable, and sets the
lower limit on the dynamic range. This noise corresponds to
2. Referenced Documents
observednoiseoftheRIdetectoronly.(Theactualnoiseofthe
LC system may be larger or smaller than the observed value,
2.1 ASTM Standards:
depending upon the method of data collection, or signal
E386Practice for Data Presentation Relating to High-
monitoringofthedetector,sinceobservednoiseisafunctionof
Resolution Nuclear Magnetic Resonance (NMR) Spec-
the frequency, speed of response and the band width of the
troscopy
recorder or other electronic circuit measuring the detector
signal.)
3. Significance and Use
4.1.2 long term noise—thisnoiseisthemaximumamplitude
3.1 Although it is possible to observe and measure each of
in RIU for random variations of the detector signal with
several characteristics of a detector under different and unique
frequencies between 6 and 60 cycles per h (0.1 and 1.0 cycles
conditions,itistheintentofthispracticethatacompletesetof
per min). It represents noise that may be mistaken for a
detector test results should be obtained under the same oper-
late-eluting peak.This noise corresponds to the observed noise
ating conditions. It should also be noted that to specify
only and may not always be present.
completely a detector’s capability, its performance should be
4.1.3 drift—the average slope of the long term noise enve-
measured at several sets of conditions within the useful range
lope expressed in RIU per hour as measured over a period of
of the detector.
1h.
4.1.4 static—refers to the noise and drift measured under
conditions of no flow.
This practice is under the jurisdiction ofASTM Committee E13 on Molecular
4.1.5 dynamic—refers to the noise and drift measured at a
Spectroscopy and Separation Science and is the direct responsibility of Subcom-
mittee E13.19 on Separation Science. flow rate of 1.0 mL/min.
Current edition approved Nov. 1, 2010. Published November 2010. Originally
4.1.6 flow sensitivity—the rate of change of signal displace-
approved in 1989. Last previous edition approved in 2005 as E1303–95(2005).
ment (in RIU) vs flow rate (in mL/min) resulting from step
DOI: 10.1520/E1303-95R10.
changes in flow rate calculated at 1 mL/min as described in
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
4.3.12.
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. 4.2 Test Conditions:
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1303 − 95 (2010)
4.2.1 Thesametestsolventmustbeusedinbothsampleand 4.3.4 Allow the chromatographic system to stabilize for at
reference cells. The test solvent used and its purity should be least 60 min without flow. The detector range, Note 2, should
specified. Water equilibrated with the laboratory atmosphere besetsuchthattheamplitudeofshorttermnoisemaybeeasily
containingminimumimpuritiesisthepreferredtestsolventfor measured. Ideally, the output should contain no filtering of the
measuring noise and drift. Water for this purpose (preferably signal. If the filtering cannot be turned off, the minimum time
purified by distillation, deionization, or reverse osmosis) constant should be set and noted in the evaluation. Manuals or
should be drawn, filtered through a 0.45-µm filter, and allowed manufacturers should be consulted to determine if time con-
to stand in a loosely covered container for several hours at stant and detector range controls are coupled, and information
ambient temperature in the laboratory in which testing is to be should be obtained to determine if they can be decoupled for
carried out. This will ensure complete equilibration of the testing. Set the recorder zero to near mid-scale. Record at least
water with the gases in the laboratory atmosphere. 1hofbaselineunderthesestaticconditions,duringwhichtime
the ambient temperature should not change by more than 2°C.
NOTE 1—It is essentially impossible to maintain a constant RI value of
de-gassedwaterandofverydilutesamplesinde-gassedwater.Thisisdue
NOTE 2—RI detectors will have one or more controls labeled
to the fact that the difference in refractive index between completely
attenuation, range, sensitivity,and scale factor.Allareusedtosetthefull
−6 3
de-gassed water and atmosphere-equilibrated water is 1.5×10 RIU.
scale range (in RIU) of an output display device such as a strip chart
Thus, small differences in the concentration of dissolved gases between
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
manufacturer’s recommendations, and this temperature should
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.
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 (2010)
4.3.6 Now mark the center (center of gravity) of each
segment over the 15-min period of the short term noise
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.
FIG. 3 Example of Plot for Calculation of Flow Sensitivity
4.3.10 Make the measurement for the dynamic long term
noise following the procedure outlined in 4.3.6.
a smooth curve connecting the points and draw a tangent at 1
4.3.11 Draw the pair of parallel lines in accordance with
mL/min (Fig. 3). Express the slope of the line as the flow
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
5.1 Descriptions of Terms Specific to this Standard:
re-equilibration,ataslowrecorderspeed.Increasetheflowrate
5.1.1 minimum detectability—that concentration of a spe-
to 1.0 mL/min and record for 15 min or more. Run at 2.0, 4.0,
cific solute in a specific solvent that gives a signal equal to
and 8.0 mL/min if the pressure flow limit of the chromato-
twice the static short-term noise.
graphic system is not exceeded.Ifnecessary,adjustthedetector
5.1.1.1 Discussion—The static short-term noise is a mea-
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).
noisewouldensurethatanyvalueoutsidethisrangewouldnot
Measure the vertical displacement between these lines, and
be noise with a confidence level of greater than 99%. Since
expressinRIU(5.2.9).Plotthesevaluesversusflowrate.Draw
4,
peak-to-peak noise is approximately five times the rms noise,
the minimum detectability defined in this practice is a more
conservative estimate. Minimum detectability, as defined in
thispractice,shouldnotbeconfusedwiththelimitofdetection
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 (2010)
linearity plot specified in 5.2.13. The linear range may be 5.2.3 Weigh out 43.6 g of glycerin (USP) and dissolve in 1
expressed in three different ways: L of the atmosphere-equilibrated purified water. This stock
solution is 50 times the concentration of the normal solution
5.1.3.1 As the ratio of the upper limit of linearity obtained
from the linearity plot, and the minimum linear concentration, (5.2.2) used for calibration and is assigned a normalized
concentration of 50.
both measured for the same test substance in the same test
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
where:
range testing.
L.R. = linear range of the detector, 5.2.5 Because atmosphere-equilibrated water is used as the
C = upper limit of linearity obtained from the l
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