荧光是直接测定的拉曼光谱中背景的最主要来源, 需要采用真实、 准确的方法消除, 以得到纯净的拉曼响应。 基线拟合消除和查找荧光贡献扣除是解决背景问题的两条思路, 目前多采用基线拟合方法, 其优点是满足用户“视觉”要求, 无需额外硬件, 但并非机理或实质上的解释, 因而难以保证数据的真实性与合理性; 查找荧光的方法, 更为真实, 但是目前提出的方法, 需要增加光源等额外设计和成本。 另外, 在实验方法上, 也有采用消荧光剂和长时间照射漂白的, 存在操作繁琐、 效率低等不足。 利用稳定体系中拉曼和荧光的时间差异解决体系中荧光问题。 在微小的时间段内, 例如几个毫秒, 激发光不会导致体系性质发生显著变化, 荧光具有寿命周期, 会随激发时间延长强度下降的“褪色”, “褪色”的强度差异可以被认为是整体荧光的一个微元; 与此同时, 由于体系组成未发生显著变化, 拉曼光对于短时间照射可以保持稳定。 利用此差异可以区分出混合信号中的荧光和拉曼光。 根据该原理, 提出了荧光褪色差分法(FBDA), 实现拉曼光谱的背景校正。 方法的主要步骤： 测量微小时刻内的多张直接拉曼光谱, 求取系列光谱的差分, 对差分值作高频滤波降噪, 可获得荧光强度微元; 然后, 多个荧光微元平均归一化后, 得到荧光强度单元。 以拉曼光谱2 000～2 500 cm-1的静默区, 即通常不会出现拉曼信号的频段为基准, 对荧光单元作逆差分, 逆差分累计值与原始光谱在此频段一致时, 得到整体荧光响应; 最终, 从原始光谱中扣除荧光成分, 完成背景扣除和基线校正。 以盐酸二甲双胍片的拉曼测量为例, 说明和讲解了所提出的原理和方法, 验证方法的有效性。 与目前效果较好的基线校正方法(不对称最小二乘和自适应迭代再加权惩罚偏最小二乘)进行了对比, 表明FBDA方法更为客观真实, FBDA的另一个优势是不需要额外的设计和成本, 所有数据都是在现有设备直接采集和完成。 需要说明的是, 微小时刻光谱差异的要求, 可以确保FBDA光谱实时性, 长时间的光谱差异, 将会影响结果的准确性; 另外, 对于光化学反应体系和其他非荧光引起的复杂背景, FBDA的适用性有待改善。

Fluorescence is the main part of background within direct Raman spectrum, and a faithful and truthful method is needed to remove it from the Raman detection, and to provide one pure Raman spectrum. There are two paths for setting background of direct spectra, i.e., baseline fitting and fluorescence finding. The baseline fitting is a currently path whose significant advantage reflects in “visual experience” without additional hardware, nevertheless, “visual” means surface and not essential, so that its results probably may be wrong or unreal. The other way attends to find out the real fluorescence within spectra, but it needs additional design and cost, such as two and more wavelengths in laser sources. Furthermore, some handle methods, reagent for suppress fluorescence or long time photo-bleaching, are insufficient operation and low efficiency. This research focus on the time difference of Raman and fluorescence to find out fluorescence from the direct spectra of a stable system. For the stable system, and within a tiny time cell, a few milliseconds or so, the excitation light does not make the system to be changed significantly and a part of fluorescence has been out of the life cycle at the same time, that is, fluorescence is fading and Raman keep stable within the time cell. So that this difference of the time cell can be treated as a fluorescence infinitesimal and used to distinguish fluorescence and Raman from mixed signal. Based on this principle, the fluorescence photo-bleaching difference approach (FBDA) has been presented to eliminate the background under direct Raman spectra. The steps of FBDA is carried out as follows: Firstly, a series of Raman spectra between tiny time cell is directly measured, and then a series differences for each time cell is computed, these differences are de-noised by low pass filtering, to get the infinitesimals of fluorescence. Secondly, these infinitesimals are mean and normalized to be a fluorescence unit. Thirdly, the total fluorescence can be summed by inverse difference of the unit, in addition, the silence area of Raman, 2 000～2 500 cm-1, which usually do not appear Raman signal, can be used as a benchmark for deciding the gross of fluorescence. Finally, the fluorescence gross deducted from the original spectrum, and the background deduction or baseline correction is completed. The paper takes an example, the Raman spectrum measuring of metformin hydrochloride tablets, to illustrate the FBDA method and its validity. The FBDA is more objective and true than others baseline correction methods, which are considered to work well, such as, ALS and airPLS. Further advances of FDBA are more convenient and less costly than the current fluorescence-finding path, because all of the data for FDBA are collected by existing instruments without any change or addition. It should be noted that the tiny time cell is a key requirement for FDBA, and tiny time cell can ensure the real-time performance of spectrum, the difference of long time would affect the accuracy of the results. In addition, the applicability of FBDA needs further development under the complex background from photochemical reaction and other non-fluorescence.