JJG 705-2014Liquid Chromatography (English PDF)

4.1 Delivery System

4.1.1 The delivery pipeline interfaces must be tight and secure, with no leakage within the specified pressure range.

4.1.2 The pump flow rate setpoint error SS and flow rate stability SR shall meet the requirements of Table 1.

4.1.3 Maximum permissible gradient error Gc. ±3%.

4.2 Column oven

4.2.1 Maximum permissible error of column oven temperature setpoint. ±2 °C.

4.2.2 Column oven temperature stability. Not greater than 1 °C/h.

4.3 Detector

The main technical indicators of the instrument's detector are shown in Table 2.

4.4 Overall instrument performance

The overall performance of the instrument is expressed by the repeatability of qualitative and quantitative measurements. The requirements are shown in Table 3.

5.1 Instrument appearance

The instrument shall have a nameplate, displaying the instrument name, model, manufacturer's name, product series number, exit-factory date, etc. Domestically produced instruments shall have a manufacturing license mark for metering instruments.

5.2 Instrument circuit system

The instrument's power cord and signal cables are securely connected. All switches, knobs, buttons function normally. Indicator lights are sensitive. The display is clear.

6.1 Verification conditions

6.1.1 Environmental conditions

6.1.1.1 The verification chamber shall be clean and dust-free, free of flammable, explosive, corrosive gases, and well-ventilated.

6.1.1.2 Room temperature (15 ~ 30) °C, with temperature changes not exceeding 3 °C during verification (for differential refractive index detectors, room temperature changes not exceeding 2 °C); relative humidity 20% ~ 85%.

6.1.1.3 The instrument shall be placed stably on the workbench, free from strong mechanical vibrations and electromagnetic interference sources, meanwhile properly grounded.

6.1.1.4 The power supply voltage is (220 ± 22) V; the frequency is (50 ± 0.5) Hz.

6.1.2 Verification equipment

6.1.2.1 Stopwatch. Minimum scale division not greater than 0.1 s.

6.1.2.2 Analytical balance. Maximum weighing capacity not less than 100 g, minimum

scale division not greater than 1 mg.

6.1.2.3 Digital thermometer. Measuring range (0 ~ 100) °C, maximum permissible error

±0.3 °C.

All the above instruments must be certified by metrological verification.

6.1.3 Certified reference materials.

Naphthalene-methanol solution reference material. Certified value 1.00 × 10-4 g/mL, expanded uncertainty < 4%, k = 2;

Naphthalene-methanol solution reference material. Certified value 1.00 × 10-7 g/mL, expanded uncertainty < 4%, k = 2;

Cholesterol solution in methanol reference material. Certified value 200 µg/mL, expanded uncertainty < 2.0%, k = 2;

Connect the detector and data processing system; set the detector wavelength to

254 nm. After power-on stabilization, inject 2% isopropanol-water solution directly into the detection cell to flush it, until the reading stabilizes. Record this value. Then, following the same method, sequentially inject a series of acetone-

2% isopropanol aqueous solutions (acetone content of 0.1%, 0.2%,..., 1.0%) into the detection cell. Record the stable response signal value corresponding to each solution. Each solution is measured 3 times. Take the arithmetic mean. Plot a standard curve, using 5 acetone solution concentrations (0.1%, 0.2%, 0.3%, 0.4%,

0.5%) and their corresponding response signal values. Find the readings at each point on the curve where the acetone solution concentration is greater than 0.5%;

compare it with the measured values at the corresponding concentration points.

The concentration at which the value differed by 5% is taken as the upper limit of detection (CH). The minimum detection concentration, which is obtained according to 6.2.2.4 c), is taken as the lower limit of detection (CL). The CH/CL ratio is taken as the linear range.

6.2.2.5 Fluorescence detector performance

a) Wavelength indication error and repeatability

For the calibration of the wavelength indication error and repeatability of the fixed-wavelength fluorescence detector, the filter in the detector needs to be removed. Referring to the method 5.3.3 in JJG 537-2006 "Verification regulation of fluorescence spectrophotometer" (see Appendix B), measure the wavelength corresponding to the maximum transmittance on a calibrated UV-Vis spectrophotometer. The difference between this wavelength and the wavelength marked on the filter is the wavelength indication error.

To determine the wavelength indication error and repeatability of the tunable wavelength fluorescence detector, connect the detector to the data processing system. Utilizing the characteristic that naphthalene has maximum fluorescence intensity at 290 nm (excitation wavelength) and 330 nm (emission wavelength), inject the 1.00 × 10-7 g/mL naphthalene-methanol standard solution into the detection cell inlet using a syringe, to flush and fill the detection cell. Adjust the excitation wavelength to 290 nm; change the emission wavelength from 325 nm to 335 nm, alternating by 1 nm every 5 ~ 10 seconds. Record the absorbance value at each wavelength (a schematic diagram of the absorbance value changes is similar to Figure 3). The difference -- between the wavelength corresponding to the highest point of the curve and the reference wavelength -- is the emission wavelength indication error. This measurement is repeated 3 times. The difference between the maximum and minimum values is the wavelength repeatability. Then, adjust the emission wavelength to the wavelength corresponding to the highest point of the measured curve; change the excitation wavelength (from 285 nm to

295 nm). Use the same method as before, to measure the indication error and repeatability of the excitation wavelength.

b) Baseline drift and baseline noise

Connect all parts of the instrument. Select a C18 column. Use 100% methanol as the mobile phase at a flow rate of 1.0 mL/min. Select the most sensitive setting.

Set the excitation wavelength to 290 nm and the emission wavelength to 330 nm.

After the instrument is warmed up and stabilized, record the baseline for 30 min.

Calculate the baseline noise, according to formula (7), based on the detector attenuation factor and the response signal value corresponding to the measured baseline peak-to-peak height, expressing it in units of the detector's own physical quantity (FU). Baseline drift is expressed as the maximum response signal value (FU/30 min) of the baseline deviation from the starting point within 30 min.

c) Minimum detection concentration

Under the chromatographic conditions in 6.2.2.5 b), after the baseline is stabilized, inject (10 ~ 20) µL of 1.00×10-7 g/mL (or 1×10-8 g/mL) naphthalene-methanol solution through the injection system; record the chromatogram; calculate the minimum detection concentration according to formula (8).

d) Linear range

Connect the detector and data processing system. Set the excitation wavelength of the detector to 290 nm and the emission wavelength to 330 nm. After the instrument stabilizes, inject 100% methanol into the detection cell to rinse it until the reading stabilizes; record this value. Then, sequentially inject 1×10-5 g/mL,

2×10-5 g/mL, 3×10-5 g/mL,..., 1×10-4 g/mL of naphthalene-methanol solutions into the cell using the same method; record the response signal value corresponding to each solution. Repeat the measurement 3 times. Take the average value. Plot a standard curve, using 5 naphthalene-methanol solution concentrations (1×10-5 to 5×10-5 g/mL) and their corresponding response signal values. Find the readings at points on the curve, where the naphthalene-methanol concentration is greater than 5×10-5 g/mL. Compare it with the measured values at the corresponding concentration points. The naphthalene-methanol solution concentration at which the two values differ by 5% is defined as the upper limit of detection (CH). The minimum detection concentration (CL) obtained in 6.2.2.5 c) is defined as the lower limit of detection. The CH/CL ratio is the linear range.

Note. The 1×10-5 g/mL, 1×10-6 g/mL, 1×10-8 g/mL naphthalene-methanol solutions used in the performance verification of the fluorescence detector can be obtained, by diluting a 1.0×10-4 g/mL naphthalene-methanol standard material.

6.2.2.6 Performance of differential refractive index detector

a) Baseline drift and baseline noise

Using a C18 column, connect all parts of the instrument. Use methanol as the mobile phase at a flow rate of 1.0 mL/min. Fill the reference cell with the mobile

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