ASTM E685-93(2013)
(Practice)Standard Practice for Testing Fixed-Wavelength Photometric Detectors Used in Liquid Chromatography
Standard Practice for Testing Fixed-Wavelength Photometric Detectors Used in Liquid Chromatography
SIGNIFICANCE AND USE
4.1 Although it is possible to observe and measure each of the several characteristics of a detector under different and unique conditions, it is the intent of this practice that a complete set of detector specifications should be obtained under the same operating conditions. It should also be noted that to completely specify a detector's capability, its performance should be measured at several sets of conditions within the useful range of the detector. The terms and tests described in this practice are sufficiently general that they may be used regardless of the ultimate operating parameters.
4.2 Linearity and response time of the recorder or other readout device used should be such that they do not distort or otherwise interfere with the performance of the detector. This requires adjusting the gain, damping, and calibration in accordance with the manufacturer's directions. If additional electronic filters or amplifiers are used between the detector and the final readout device, their characteristics should also first be established.
SCOPE
1.1 This practice is intended to serve as a guide for the testing of the performance of a photometric detector (PD) used as the detection component of a liquid-chromatographic (LC) system operating at one or more fixed wavelengths in the range 210 to 800 nm. Measurements are made at 254 nm, if possible, and are optional at other wavelengths.
1.2 This practice is intended to describe the performance of the detector both independently of the chromatographic system (static conditions) and with flowing solvent (dynamic conditions).
1.3 For general liquid chromatographic procedures, consult Refs (1-9).2
1.4 For general information concerning the principles, construction, operation, and evaluation of liquid-chromatography detectors, see Refs (10 and 11) in addition to the sections devoted to detectors in Refs (1-7).
1.5 This standard does not purport to address all of the safety problems, 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.
General Information
Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E685 − 93 (Reapproved 2013)
Standard Practice for
Testing Fixed-Wavelength Photometric Detectors Used in
Liquid Chromatography
This standard is issued under the fixed designation E685; 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. Terminology
1.1 This practice is intended to serve as a guide for the 3.1 Definitions:
testingoftheperformanceofaphotometricdetector(PD)used 3.1.1 absorbance calibration, n—the procedure that verifies
as the detection component of a liquid-chromatographic (LC) that the absorbance scale is correct within 65%.
systemoperatingatoneormorefixedwavelengthsintherange
3.1.2 drift, n—the average slope of the noise envelope
210to800nm.Measurementsaremadeat254nm,ifpossible,
expressed in absorbance units per hour (AU/h) as measured
and are optional at other wavelengths.
over a period of 1 h.
1.2 This practice is intended to describe the performance of
3.1.3 dynamic, n—under conditions of a flow rate of 1.0
thedetectorbothindependentlyofthechromatographicsystem
mL/min.
(static conditions) and with flowing solvent (dynamic condi-
3.1.4 linear range, n— of a PD, the range of concentrations
tions).
of a test substance in a mobile phase over which the response
1.3 For general liquid chromatographic procedures, consult ofthedetectorisconstanttowithin5%asdeterminedfromthe
Refs (1-9). linearity plot specified below and illustrated in Fig. 1. The
linear range should be expressed as the ratio of the highest
1.4 For general information concerning the principles,
concentration to the minimum detectable concentration or the
construction, operation, and evaluation of liquid-
lowest linear concentration, whichever is greatest.
chromatography detectors, see Refs (10 and 11) in addition to
3.1.5 long-term noise, n—the maximum amplitude in AU
the sections devoted to detectors in Refs (1-7).
for all random variations of the detector signal of frequencies
1.5 This standard does not purport to address all of the
between6and60cyclesperhour(0.1and1.0cyclespermin).
safety problems, if any, associated with its use. It is the
3.1.5.1 Discussion—Itrepresentsnoisethatcanbemistaken
responsibility of the user of this standard to establish appro-
for a late-eluting peak. This noise corresponds to the observed
priate safety and health practices and determine the applica-
noise only and may not always be present.
bility of regulatory limitations prior to use.
3.1.6 minimum detectability, n—of a PD, that concentration
ofaspecificsoluteinaspecificsolventthatresultsinadetector
2. Referenced Documents
response corresponding to twice the static short-term noise.
2.1 ASTM Standards:
3.1.7 response time (speed of output), n—the detector, the
E275PracticeforDescribingandMeasuringPerformanceof
timerequiredforthedetectoroutputtochangefrom10to90%
Ultraviolet and Visible Spectrophotometers
of the new equilibrium value when the composition of the
E682Practice for Liquid Chromatography Terms and Rela-
mobilephaseischangedinastepwisemanner,withinthelinear
tionships
range of the detector.
3.1.7.1 Discussion—Because the detector volume is very
small and the transport rate is not diffusion dependent, the
This practice is under the jurisdiction ofASTM Committee E13 on Molecular
response time is generally fast enough to be unimportant. It is
Spectroscopy and Separation Science and is the direct responsibility of Subcom-
generally comparable to the response time of the recorder and
mittee E13.19 on Separation Science.
Current edition approved Jan. 1, 2013. Published January 13. Originally
dependent on the response time of the detector electrometer
approved in 1979. Last previous edition approved in 2005 as E685–93(2005).
and on the recorder amplifier. Factors that affect the observed
DOI: 10.1520/E0685-93R13.
response time include the true detector response time, elec-
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
tronic filtering, and system band-broadening.
this practice.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
3.1.8 short-term noise, n—the maximum amplitude, peak to
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
peak, inAU for all random variations of the detector signal of
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. a frequency greater than one cycle per minute.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E685 − 93 (2013)
that there is ozone in the air. Protect the entire system from
temperature fluctuations because these will lead to detectable
drift.
5.1.1 The detector should be located at the test site and
turned on at least 24 h before the start of testing. Insufficient
warm-upmayresultindriftinexcessoftheactualvalueforthe
detector.
5.2 Methods of Measurement:
5.2.1 Connect a suitable device (Note 1) between the pump
and the detector to provide at least 75 kPa (500 psi) back
pressure at 1.0 mL/min flow of methanol. Connect a short
length(about100mm)of0.25-mm(0.01-in.)internal-diameter
stainless steel tubing to the outlet tube of the detector to retard
bubble formation. Connect the recorder to the proper detector
output channels.
NOTE 1—Suggested devices include (a)2to4mof 0.1-mm (0.004-in.)
internal-diameter stainless steel tubing, (b) about 250 mm of 0.25 to
0.5-mm (0.01 to 0.02-in.) internal-diameter stainless steel tubing crimped
with pliers or cutters, or (c) a constant back-pressure valve located
between the pump and the injector.
FIG. 1 Example of a Linearity Plot for a Photometric Detector
5.2.2 Repeatedly rinse the reservoir and chromatographic
system, including the detector, with degassed methanol to
remove from the system all other solvents, any soluble
3.1.8.1 Discussion—Itdeterminesthesmallestsignaldetect-
material, and any entrained gasses. Fill the reservoir with
able by a PD, limits the precision attainable in quantitation of
methanolandpumpthissolventthroughthesystemforatleast
trace-level samples, and sets the lower limit on linearity. This
30 min to complete the system cleanup.
noise corresponds to the observed noise only.
5.2.3 Air or nitrogen is used in the reference cell, if any.
3.1.9 static, n—under conditions of no flow.
Ensure that the cell is clean, free of dust, and completely dry.
5.2.4 To perform the static test, cease pumping and allow
4. Significance and Use
thechromatographicsystemtostabilizeforatleast1hatroom
temperature without flow. Set the attenuator at maximum
4.1 Although it is possible to observe and measure each of
sensitivity (lowest attenuation), that is, the setting for the
the several characteristics of a detector under different and
smallest value of absorbance units full-scale (AUFS). Adjust
unique conditions, it is the intent of this practice that a
the response time as close as possible to 2 s for a PD that has
complete set of detector specifications should be obtained
a variable response time (Note 2). Record the response time
under the same operating conditions. It should also be noted
used.Adjustthedetectoroutputtonearmidscaleonthereadout
that to completely specify a detector’s capability, its perfor-
device. Record at least1hof detector signal under these
mance should be measured at several sets of conditions within
conditions, during which time the ambient temperature should
the useful range of the detector. The terms and tests described
in this practice are sufficiently general that they may be used not change by more than 2°C.
regardless of the ultimate operating parameters.
NOTE2—Timeconstantisconvertedtoresponsetimebymultiplyingby
the factor 2.2. The effect of electronic filtering on observed noise may be
4.2 Linearity and response time of the recorder or other
studied by repeating the noise measurements for a series of response-time
readout device used should be such that they do not distort or
settings.
otherwise interfere with the performance of the detector. This
5.2.5 Draw pairs of parallel lines, each pair corresponding
requires adjusting the gain, damping, and calibration in accor-
to between 0.5 and 1 min in length, to form an envelope of all
dance with the manufacturer’s directions. If additional elec-
observed random variations over any 15-min period (see Fig.
tronicfiltersoramplifiersareusedbetweenthedetectorandthe
2). Draw the parallel lines in such a way as to minimize the
final readout device, their characteristics should also first be
distance between them. Measure the vertical distance, in AU,
established.
between the lines. Calculate the average value over all the
5. Noise and Drift segments.Dividethisvaluebythecelllengthincentimetresto
obtain the static short-term noise.
5.1 Test Conditions—Pure, degassed methanol of suitable
4 5.2.6 Nowmarkthecenterofeachsegmentoverthe15-min
grade shallbeusedinthesamplecell.Airornitrogenshallbe
period of the static short-term noise measurement. Draw a
used in the reference cell if there is one. Nitrogen is preferred
series of parallel lines encompassing these centers, each pair
where the presence of high-voltage equipment makes it likely
correspondingto10mininlength,andchoosethatpairoflines
whose vertical distance apart is greatest (see Fig. 2). Divide
this distance inAU by the cell length in centimetres to obtain
Distilled-in-glass or liquid-chromatography grade. Complete freedom from
particles may require filtration, for example, through a 0.45-µm membrane filter. the static long-term noise.
E685 − 93 (2013)
FIG. 2 Example for the Measurement of the Noise and Drift of a PD (Chart Recorder Output).
5.2.7 Draw the pair of parallel lines that minimizes the 5.2.9 Draw pairs of parallel lines, measure the vertical
vertical distance separating these lines over the 1 h of mea- distances, and calculate the dynamic short-term noise follow-
surement (see Fig. 2).The slope of either line is the static drift ing the procedure of 5.2.5.
expressed in AU/h. 5.2.10 Make the measurement for the dynamic long-term
5.2.8 Set the pump to deliver 1.0 mL/min under the same noise following the procedure outlined in 5.2.6.
conditions of tubing, solvent, and temperature as in 5.2.1 5.2.11 Draw the pair of parallel lines as directed in 5.2.7.
through5.2.3.Allow15minforthesystemtostabilize.Record The slope of these lines is the dynamic drift.
at least1hof signal under these flowing conditions, during 5.2.12 The actual noise of the system may be larger or
which time the ambient temperature should not change by smaller than the observed values, depending upon the method
more than 2°C. of data collection, or signal monitoring of the detector, since
E685 − 93 (2013)
observed noise is a function of the frequency, speed of slope 3MW
ε 5 (1)
response, and bandwidth of the readout device. b
where:
6. Minimum Detectability, Linear Range, and
slope = the slope of the linear portion of the plot,AU·µl/µg,
Calibration
MW = molecular weight, g/mole, and
6.1 Methods of Measurement—For the determination of the
b = nominal cell length, cm, as specified by the
linearrangeofaPD, (12)foraspecificsubstance,theresponse
manufacturer.
to that test substance must be determined. The following
Compare the value of ε obtained with an experimentally
procedure is designed to provide a worst-case procedure.
determined value or one from the literature (Note 3). Should
6.1.1 Dissolve in methanol a suitable compound with an
the values differ by more than 5%, the PD may require
ultraviolet spectral absorbance that changes rapidly at the
adjustment. Consult the manufacturer’s directions.
wavelength of interest. Choose a concentration that is ex-
NOTE 3—For example, the values of molar absorptivity for uracil in
pected to exceed the linear range, typically to give an absor-
3 3
methanol are 7.7×10 at 254 nm and 1.42×10 at 280 nm; for potassium
bance above 2AU. Dilute the solution accurately in a series to
dichromate in 0.01 N sulfuric acid they are 4.22×10 at 254 nm and
coverthelinearrange,thatis,downtotheminimumdetectable
3.60×10 at 280 nm.
concentration. Rinse the sample cell with methanol and zero
7. Response Time
the detector with methanol in the cell. Rinse the cell with the
solution of lowest concentration until a stable reading is
7.1 The response time of the detector may become signifi-
obtained; usually rinsing the cell with 1 mL is sufficient.
cant when a short micro-particle column and a high-speed
Record the detector output. After rinsing the syringe thor-
recorderareused.Also,itispossible,byusinganintentionally
oughly with the next more concentrated solution, fill the cell
slow response time, to reduce the observed noise and hence
withthesolutionfromeachdilutioninturn.Obtainaminimum
increase the apparent linear range. Although this would have
of five on-scale measurements. Measure under static condi-
little effect on broad peaks, the signal from narrow peaks
tions.
would be significantly degraded. Measure at the highest and
6.1.2 Calculate the ratio of detector response (AU) to
lowest values of the electronic filter if it is variable.
concentration (µg/mL) for each solution and plot these ratios
7.2 Method of Measurement:
versus log concentration (see Fig. 1). The region of linearity
7.2.1 The composition of the mobile phase is changed in a
will define a horizontal line of constant response ratio. At
stepwise manner and the output signal is recorded on the
higher concentrations, there will typically be a negative devia-
highest-speed device available. If the recorder has a response
tion from linearity, while at lower concentrations there may be
timenotsignificantlyfasterthanthedetector,onlytheresponse
deviation in either direction. Draw horizontal lines 5% above
time of the detector-recorder combination will be obtained, as
and below the line of constant response ratio. The upper limit
it would be when the combination is used to record chromato-
of linearity is the concentration at which the line of measured
grams.
response ratio intersects one of the 5% bracketing lines at the
7.2.2 Set a flow rate of 2.0 mL/min.
highconcentrationend.Thelowerlimitoflinearityiseitherthe
7.2.3 A stepwise change may be obtained by means of a
minimum detectable concentration (see 6.1.3) or the concen-
sample valve equipped with a 1-mL sample loop (or a loop
tration at which the line of measured response ratio intersects
having at least four times the total volume from detector inlet
one of the bracketin
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.