基于光源切换的发光二极管诱导荧光检测器的研制与评价

Development and Evaluation of a Light-emitting Diode Induced Fluorescence Detector Based on Light Source Switching

荧光检测器具有灵敏度高、选择性好等特点,是高效液相色谱法(HPLC)常用的检测器之一。该文研制了一款基于共聚焦光学构型的多波长发光二极管(LED)诱导荧光检测器,可实现多种物质的检测。通过电机多重校准定位与光源滑动定位技术实现多光源的精准定位,使用共用发射光路简化仪器结构并降低了仪器成本。针对LED光源切换机构进行3万个周期连续切换的可靠性测试,测得激光器的靶点位置误差在±0.2 mm以内,实现了LED光源定位的准确性和良好重复性,解决了传统荧光检测器结构复杂、价格高,以及发光二极管(LED)单波长诱导荧光检测器专用性强、应用范围窄等问题。以甲醇为流动相,使用C_(18)色谱柱对3个光源进行基线考察,结果表明:研制的检测器在340、365、385 nm激发光源下,基线噪声低于5.0×10^(-4) FU,漂移小于5.0×10^(-3) FU/h。以真菌毒素和苯并(α)芘为探针,对其线性和灵敏度进行评价。分别采用乙腈-水-乙酸(96∶102∶2,体积比)、甲醇-乙腈-水(20∶20∶60)、乙腈-水(90∶10)作为检测赭曲霉素A、黄曲霉毒素B1、苯并(α)芘的流动相,激发波长分别为340、365、385 nm。测得赭曲霉素A、黄曲霉毒素B1、苯并(α)芘分别在1.0~50.0、0.1~20.0、0.5~20.0 ng/mL质量浓度范围内呈良好的线性关系(r^(2)≥0.9999),检出限分别为0.06、0.01、0.02 ng/mL。对该检测器的重复性进行考察,结果表明3个光源对样品响应值的RSD(n=11)均小于1.0%。以340 nm光源为测试条件,显示检测器在震动、高温和低温环境下的基本性能均未发生明显变化。与市售进口的通用型荧光检测器相比,二者在基线噪声、漂移和灵敏度方面结果相当。该文研制的检测器结构简单、波长覆盖范围宽、性能良好,在真菌毒素和苯并(α)芘检测中具有良好的应用前景。

Fluorescence detector has the characteristics of high sensitivity and good selectivity.It is one of the most commonly used detectors in high performance liquid chromatography(HPLC).In this paper,a multi-wavelength light-emitting diode(LED)induced fluorescence detector was developed based on a confocal optical configuration,which could be applied in detection of variety of substances.The motor multiple calibration positioning technique and light source sliding positioning technique were used to achieve the precise positioning of multiple light sources.And a shared emission optical path was used to simplify the instrument structure and reduce the cost of the instrument.The reliabil⁃ity test on the continuous switching of LED light source for 30000 cycles showed that the target posi⁃tion errors of the laser ranged within±0.2 mm,which realized the accuracy and good repeatability of LED light source positioning,solving the problems of traditional fluorescence detector with complex structure and high price,and LED single wavelength induced fluorescence detector with strong speci⁃ficity and narrow application range.A mobile phase of methanol and a C_(18) chromatographic column were used to investigate the baselines of three light sources.Results showed that the developed detec⁃tor,under excitation light sources of 340,365 and 385 nm,had baseline noises less than 5.0×10^(-4) FU and a drifts less than 5.0×10^(-3) FU/h.Furthermore,mycotoxins and benzo(α)pyrene were used as probes to evaluate the linearity and sensitivity.Meanwhile,acetonitrile-water-acetic acid(96∶102∶2,by volume),methanol-acetonitrile-water(20∶20∶60)and acetonitrile-water(90∶10)were used as mobile phases for the analysis of ochratoxin A,aflatoxin B1 and benzo(α)py⁃rene,respectively.The excitation wavelengths of the detector were set at 340,365 and 385 nm,respectively.The concentrations of ochratoxin A,aflatoxin B1 and benzo(α)pyrene showed good lin⁃earities(r^(2)≥0.9999)in the ranges of 1.0-50.0 ng/mL,0.1-20.0 ng/mL and 0.5-20.0 ng/mL,respectively.The limits of detection were 0.06,0.01 and 0.02 ng/mL,respectively.The re⁃peatability of this detector was investigated,and the result showed that the RSDs(n=11)for sample response values of the three light sources were all less than 1.0%.While using the 340 nm light source as the test condition,the basic performance of the detector under vibration,high temperature and low temperature environments did not changed significantly.The tested results of the detector were comparable in terms of baseline noise,drift and sensitivity,compared with those of the com⁃mercially available imported general-purpose fluorescence detectors.With the advantages of simple structure,wide wavelength coverage and good performance,the developed detector showed good ap⁃plication prospects in the detection of mycotoxins and benzo(α)pyrene.