ASTM E1303-95(2017)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E1303 − 95 (Reapproved 2017)
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 conditions,itistheintentofthispracticethatacompletesetof
detector test results should be obtained under the same oper-
1.1 This practice covers tests used to evaluate the perfor-
ating conditions. It should also be noted that to specify
mance and to list certain descriptive specifications of a
completely a detector’s capability, its performance should be
refractive index (RI) detector used as the detection component
measured at several sets of conditions within the useful range
of a liquid chromatographic (LC) system.
of the detector.
1.2 This practice is intended to describe the performance of
3.2 Theobjectiveofthispracticeistotestthedetectorunder
the detector both independent of the chromatographic system
specified conditions and in a configuration without an LC
(static conditions, without flowing solvent) and with flowing
column. This is a separation independent test. In certain
solvent (dynamic conditions).
circumstances it might also be necessary to test the detector in
1.3 The values stated in SI units are to be regarded as the
the separation mode with an LC column in the system, and the
standard.
appropriate concerns are also mentioned. The terms and tests
1.4 This standard does not purport to address all of the
described in this practice are sufficiently general so that they
safety concerns, if any, associated with its use. It is the may be adapted for use at whatever conditions may be chosen
responsibility of the user of this standard to establish appro-
for other reasons.
priate safety, health, and environmental practices and deter-
4. Noise, Drift, and Flow Sensitivity
mine the applicability of regulatory limitations prior to use.
1.5 This international standard was developed in accor- 4.1 Descriptions of Terms Specific to This Standard:
dance with internationally recognized principles on standard- 4.1.1 short term noise—this noise is the mean amplitude in
ization established in the Decision on Principles for the refractive index units (RIU) for random variations of the
Development of International Standards, Guides and Recom- detector signal having a frequency of one or more cycles per
mendations issued by the World Trade Organization Technical minute. Short term noise limits the smallest signal detectable
Barriers to Trade (TBT) Committee. 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
2.1 ASTM Standards: LC system may be larger or smaller than the observed value,
E386Practice for Data Presentation Relating to High- depending upon the method of data collection, or signal
Resolution Nuclear Magnetic Resonance (NMR) Spec- monitoringofthedetector,sinceobservednoiseisafunctionof
troscopy (Withdrawn 2015) the frequency, speed of response and the band width of the
recorder or other electronic circuit measuring the detector
3. Significance and Use
signal.)
3.1 Although it is possible to observe and measure each of 4.1.2 long term noise—thisnoiseisthemaximumamplitude
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
per min). It represents noise that may be mistaken for a
This practice is under the jurisdiction ofASTM Committee E13 on Molecular
late-eluting peak.This noise corresponds to the observed noise
Spectroscopy and Separation Science and is the direct responsibility of Subcom-
mittee E13.19 on Separation Science.
only and may not always be present.
Current edition approved Oct. 1, 2017. Published October 2017. Originally
4.1.3 drift—the average slope of the long term noise enve-
approved in 1989. Last previous edition approved in 2010 as E1303–95(2010).
lope expressed in RIU per hour as measured over a period of
DOI: 10.1520/E1303-95R17.
1h.
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
4.1.4 static—refers to the noise and drift measured under
Standards volume information, refer to the standard’s Document Summary page on
conditions of no flow.
the ASTM website.
3 4.1.5 dynamic—refers to the noise and drift measured at a
The last approved version of this historical standard is referenced on
www.astm.org. flow rate of 1.0 mL/min.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1303 − 95 (2017)
4.1.6 flow sensitivity—the rate of change of signal displace- 4.3.3 Thoroughly flush the reference cell with the same
ment (in RIU) vs flow rate (in mL/min) resulting from step solvent; keep the reference cell static.
changes in flow rate calculated at 1 mL/min as described in
4.3.3.1 It may be necessary to flush both sample and
4.3.12. reference cells with an intermediate solvent (such as methanol
or acetone), if the solvent previously used in the system is
4.2 Test Conditions:
immiscible with the test solvent.
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
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
−6 4
de-gassed water and atmosphere-equilibrated water is 1.5×10 RIU.
attenuation, range, sensitivity,and scale factor.Allareusedtosetthefull
Thus, small differences in the concentration of dissolved gases between
scale range (in RIU) of an output display device such as a strip chart
the sample and the trapped reference can lead to significant errors in
recorder.
measurement of solutions where the expected difference in RI due to
−6
4.3.5 Draw pairs of parallel lines, each between ⁄2 to 1 min
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, in length, to form an envelope of all observed random
atmosphere-equilibrated water (5.2.1) is recommended as the solvent for
variations over any 15-min period (Fig. 1). Draw the parallel
determining linearity and minimum detectability (Section 5).
lines in such a way as to minimize the distance between them.
4.2.2 The detector should be located at the test site and
Measure the distance perpendicular to the time axis between
switched on at least 24 h prior to the start of testing. Some
the parallel lines. Convert this value to RIU (5.2.9). Calculate
detectors provide an oven to thermostat the optics assembly.
the mean value over all the segments; this value is the static
Theovenshouldbesetatasuitabletemperature,followingthe
short term noise.
manufacturer’s recommendations, and this temperature should
4.3.6 Now mark the center (center of gravity) of each
be noted and maintained throughout the test procedures.
segment over the 15-min period of the short term noise
4.2.3 Linearity and speed of response of the recorder or
measurement. Draw a series of parallel lines to these centers,
other data acquisition device used should be such that it does
each 10 min in length (Fig. 1), and choose that pair of lines
not distort or otherwise interfere with the performance of the
whose distance apart perpendicular to the time axis is greatest.
detector. Ifadditionalamplifiersareusedbetweenthedetector
This distance is the static long term noise.
and the final readout device, their characteristics should also
4.3.7 Draw the pair of parallel lines, over the1hof
first be established.
measurement, that minimizes the distance perpendicular to the
time axis between the parallel lines. The slope of either line,
4.3 Methods of Measurement:
measured in RIU/h, is the static drift.
4.3.1 Connecta1m (39.37 in.) length of clean, dry,
4.3.8 Set the solvent delivery system to a flow rate that has
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 previously been shown to deliver 1.0 mL/min under the same
conditions of capillary tubing, solvent, and temperature.Allow
straight or coiled to minimize the space requirement. The
tubingshouldterminateinstandardlowdeadvolumefittingsto at least 15 min to stabilize. Set the recorder zero near
mid-scale. Record at least1hof baseline under these flowing
connect with the detector and to the pump. Commercial
chromatographs may already contain some capillary tubing to conditions, during which time the ambient temperature should
not change by more than 2°C.
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 4.3.9 Drawpairsofparallellines,measuretheperpendicular
length;ifsignificantlywider,itshouldbereplacedforthistest. distances, and calculate the dynamic short term noise, in the
4.3.2 Repeatedly rinse the reservoir and chromatographic manner described in 4.3.5 for the static short term noise.
system,includingthedetector,withthetestsolventpreparedas 4.3.10 Make the measurement for the dynamic long term
described in 4.2.1, until all previous solvent is removed from
noise following the procedure outlined in 4.3.6.
the system. Fill the reservoir with the test solvent.
4.3.11 Draw the pair of parallel lines in accordance with
4.3.7. The slope of this line is the dynamic drift.
4.3.12 Stopthechromatographicflow.Allowatleast15min
Munk, M. N., Liquid Chromatography Detectors, (T. M. Vickrey, Ed.), Marcel
for re-equilibration. Set the recorder at about 5% of full scale
Dekker, New York and Basel, 1983, pp. 165–204.
and leave the detector range setting at the value used for the
Bonsall, R. B., “The Chromatography Slave—The Recorder,” Journal of Gas
Chromatography, Vol 2, 1964, pp. 277–284. noise measurements. Set the solvent delivery system at a flow
E1303 − 95 (2017)
FIG. 2 Example for the Measurement of Flow Sensitivity
FIG. 1 Examples for the Measurement of Short Term Noise, Long
Term Noise and Drift
rate of 0.5 mL/min. Run for 15 min, or more if necessary for
re-equilibration,ataslowrecorderspeed.Increasetheflowrate
to 1.0 mL/min and record for 15 min or more. Run at 2.0, 4.0,
and 8.0 mL/min if the pressure flow limit of the chromato-
graphic system is not exceeded.Ifnecessary,adjustthedetector
FIG. 3 Example of Plot for Calculation of Flow Sensitivity
range to maintain an on-scale response.
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
peak-to-peak noise is approximately five times the rms
a smooth curve connecting the points and draw a tangent at
5,6
noise, the minimum detectability defined in this practice is a
1mL⁄min (Fig. 3). Express the slope of the line as the flow
more conservative estimate. Minimum detectability, as defined
sensitivityinRIUmin/mL.Itispreferredtogivethenumerical
in this practice, should not be confused with the limit of
value and show the plot as well.
detection in an analytical method using a refractive index
detector.
5. Minimum Detectability, Linear Range, Dynamic
5.1.2 sensitivity (response factor)—the signal output per
Range, and Calibration
unit concentration of the test substance in the test solvent, in
5.1 Descriptions of Terms Specific to this Standard:
accordance with the following relationship:
5.1.1 minimum detectability—that concentration of a spe-
S 5 R/C (1)
cific solute in a specific solvent that gives a signal equal to
twice the static short-term noise.
5.1.1.1 Discussion—The static short-term noise is a mea-
Blair, E. J., Introduction to Chemical Instrumentation, McGraw-Hill, New
surement of peak-to-peak noise.Astatistical approach to noise York, NY, 1962, and Practice E386.
E1303 − 95 (2017)
where: is chosen as the normal solution and defined to have the value
of 1. Note the detector range setting at which the normal
S = sensitivity (response factor), RIU·L/g,
solution produces a near full scale deflection and term this
R = measured detector response, RIU, and
C = concentration of the test substance in the test solvent normal range setting.
5.2.3 Weigh out 43.6 g of glycerin (USP) and dissolve in
g/L.
1L of the atmosphere-equilibrated purified water. This stock
5.1.3 linear range—the range of concentrations of the test
solution is 50 times the concentration of the normal solution
substance in the test solvent, over which the sensitivity of the
(5.2.2) used for calibration and is assigned a normalized
detector is constant to with 5% as determined from the
concentration of 50.
linearity plot specified in 5.2.13. The linear range may be
5.2.4 Serially dilute the stock solution (5.2.2) to 0.01
expressed in three different ways:
relative concentration according to Table 1. Use the stock
5.1.3.1 As the ratio of the upper limit of linearity obtained
solution and the diluted solutions for linearity and dynamic
from the linearity plot, and the minimum linear concentration,
range testing.
both measured for the same test substance in the same test
5.2.5 Because atmosphere-equilibrated water is used as the
solvent as follows:
mobile phase and sample diluent for this procedure, it is
L.R. 5 C /C (2)
advisable to apply a slight back pressure to the sample cell to
max min
prevent outgassing in the cell. This may be safely achieved by
where:
placing the solvent waste container on a shelf above the
L.R. = linear range of the detector,
detector. Avoid backpressure >690 KPa (100 psi) to prevent
C = upper limit of linearity obtained from the linearity
max
cell rupture.
plot, g/L, and
5.2.6 Measure the detector response under static conditions
C = minimum linear concentration, g/L, as defined in
min
for each of the solutions prepared in 5.2.4. Introduce the
5.2.13.1, the minimum linear concentration should
solutions conveniently using a liquid chromatography solvent
also be stated.
delivery system and an injector equipped with a 5-mL sample
5.1.3.2 Bygivingtheminimumlinearconcentrationandthe
loop. (Twenty feet of 1.02-mm (0.04-in.) inside diameter
−3
upper limit of linearity (for example, from 8.72×10 g/L to
stainless steel tubing has a volume of about 5 mL.) For each
−1
8.72×10 g/L).
measurement, pump atmosphere-equilibrated water through
5.1.3.3 Bygivingthelinearityplotitself,withtheminimum the sample cell until the baseline is stable. Stop the flow and
linear concentration and the upper limit of linearity indicated
note the position of the baseline on the chart. Load the injector
on the plot.
with the test solution and pump the solution into the sample
5.1.4 Dynamic Range—That range of concentrations of the cell. The recorded signal on the chart will change. When the
test substance in the test solvent, over which an incremental recorded signal for the test solution has stabilized, again stop
change in concentration produces an incremental change in theflowandnotethepositionofthesignalonthechart.Adjust
detector signal. The upper limit is the highest concentration at the detector range so that the distance from the water baseline
to the test solution signal can be easily measured. Finally,
which a slight further increase in concentration will give an
observable increase in detector signal. The dynamic range is restart the flow to flush the test solution from the sample cell.
Repeat this process 3 to 5 times for each test solution.
the ratio of these upper and lower limits.The dynamic range is
largerthanorequaltothelinearrange,butobviouslycannotbe Dependingontheconfigurationofthedetector,asecondpump
may be required to deliver water to the reference cell. As an
smaller.Thedynamicrangemaybeexpressedinthreedifferent
ways: alternative, fill and flush the sample cell manually using a
10-mL syringe to deliver 5 to 10 mL of solution.
5.1.4.1 As the ratio of the upper limit of dynamic range to
the minimum detectability. The minimum detectability must
also be stated.
Scott, R. P. W., “Liquid Chromatography Detectors,” 2nd edition, Journal of
5.1.4.2 By giving the minimum detectability and the upper Chromatography Library, Vol 33, Elsevier Scientific Publishing Co., Amsterdam,
−3
1986. This reference is given for general reading.
limit of dynamic range (for example, from 2.9×10 g/L to
17.4 g/L).
TABLE 1 Concentrations of Test Solutions
5.1.4.3 By giving the dynamic plot itself with the minimum
Relative Actual Concentration Theoretical RI
detectability indicated on the plot.
Concentration (g/L) Difference (RIU)
−3
5.2 Method of Measurement:
50.0 43.6 5 × 10
−3
20.0 17.4 2 × 10
5.2.1 Waterdrawnforthemobilephaseandsampledilution
−3
10.0 8.72 1 × 10
(preferably purified by distillation, deionization, or reverse
−4
5.0 4.36 5×10
−4
osmosis) should be allowed to stand for several hours at the
2.0 1.74 2×10
−1 −4
1.0 8.72×10 1×10
temperature of the room in which the testing is to be carried
−1 −5
0.5 4.36×10 5×10
out. This will ensure complete equilibration of the water with
−1 −5
0.2 1.74×10 2×10
−1 −5
the gases in the laboratory atmosphere (refer to Note 1).
0.1 8.72×10 1×10
−2 −6
−4
0.05 4.36 × 10 5×10
5.2.2 Because a 1×10 RIU difference is near the middle
−2 −6
0.02 1.74 × 10 2×10
of the operating range for most refractive index detectors, the −3 −6
0.01 8.72 × 10 1×10
−4
solution that gives 1×10 RIU when measured against water
E1303 − 95 (2017)
5.2.7 Thedetectorresponseisthedistanceincentimetreson 5.2.
...