主题：【线上讲座之十】：分光光度计的检测器（答疑结束）

原文由 jianbiao1234 发表:
我不知道这样的讨论有没有偏离主题，不过我猜很多人都已经看不懂了，反正我是云里雾里了。毕竟我们只是使用仪器而已，有很多东西没必要学得跟你们检修仪器人一样精通！所以，能不能拜托讲点浅显的，当然也有可能是我无知者无惧，提的建议太浅显了！

您好，CID和CCD其实都有比较形象的比喻。如果再浅一点，那一般的讲分析仪器的书上一般都有。我再把一般书上都有的东西发到这里，意义就没有了。

有没有象分光光度计那样既可测试吸光度，又可象荧光分光光度计那样测荧光强度的二合一仪器？
当然荧光分光光度计的检测限低得多，有同样能满足两种测试模式和要求的仪器吗？
有些分光光度计也可以在光路的90度位置附加检测器，但是检测器前没有分光，光源也不行，软件好像也没支持荧光测试。

这个问题发在这儿，我想问题应该与检测器有关，另外也想借nemoium专家的人气，沾点光。呵呵。

原文由 tutm 发表:
有没有象分光光度计那样既可测试吸光度，又可象荧光分光光度计那样测荧光强度的二合一仪器？
当然荧光分光光度计的检测限低得多，有同样能满足两种测试模式和要求的仪器吗？
有些分光光度计也可以在光路的90度位置附加检测器，但是检测器前没有分光，光源也不行，软件好像也没支持荧光测试。

这个问题发在这儿，我想问题应该与检测器有关，另外也想借nemoium专家的人气，沾点光。呵呵。

都要结贴了，还往这里发，看来是要深挖洞，广藏宝了。这个贴还是发到分子荧光论坛最好了。毕竟分光光度计大家都比较了解了。

发这里就是金子也变铁疙瘩了。

没关系，如果结贴后没有理想的建议，我再往那儿发。现在别让这儿停顿下来。而且，看这一期的应该搞仪器的较多，了解这方面的版友更多些，我想机会也会多些。

尊敬的Nemoium老师，就检测器而言，其实国外分光光度计本身最近十年来真的是没有什么大的突破和改进了，因为如果没有新技术出现的话，分光光度计已经是非常非常成熟的产品了。
相信以下一份很久以前的介绍应该会对您有所帮助。

当然国内除了普析分光光度计硬件稍好外，国内整体还没有达到国外10年前的水平，悲哀呀。

Single Beam:
The spectrophotometer uses only one beam to measure the absorbance or %T of a sample. The blank is placed in the sample compartment and the instrument is zeroed. The sample is then placed in the instrument and a measurement is made. On scanning instruments the background of the blank is stored and is subsequently subtracted from the sample by means of a microprocessor in the instrument. The zero point measurement is not repeated in a single beam instrument until the blank is again put in the instrument and rezeroed. This is the reason there is four times more drift in single beam instruments.
Background correction is not as precise on a single beam instrument as on a double beam instrument due to the subtraction process. As the background level increases, the blank becomes a larger and larger proportion of the total signal. This decreases the accuracy of the correction process. In a double beam instrument the ratio recording design automatically cancels the difference in the two optical paths and then the background correction algorithm is applied to the data. The background is maintained a proportion of the total signal by the ratio recording system. The reason for building a single beam instrument is that there are fewer optics and no chopper thus reducing the cost to manufacturer. Customer doing kinetics sacrifice stability which is critical to their experiment. Reference beam attenuation capabilities are reduced as well.

    Terms to denote single beam
        Stable Beam Technology     (Beckman)
        Temporal Double Beam      (Gilford)
        Diode Array         (Hewlett-Packard)

Dual Beam:
The beam is split, a portion going thru the blank and another portion going thru the sample. This may or may not be a 50:50 split. Some manufactures may only split 10% of the beam thru the blank and pass 90% thru the sample. The beams are ratioed electronically. Dual beam instruments measure the dark current of the system at power on and never recheck it( The Lambda 2 is an exception in that it measures the dark current every ten minutes while the instrument is not being used). This will increase the drift of the instrument. The advantage of a dual beam instrument is that one is able to monitor the drift of the instrument and correct of any change as in a double beam instrument. There is no chopper thus reducing the total cost of the instrument. A possible disadvantage is, by splitting the beam, reduces the energy thru-put of the system by up to fifty percent. This can be compensated for if very large optics are used as in the Lambda 2. However; if the optics remain the same size as a conventional instrument, the S/N is cut in half. This reduces the S/N ratio and limits the lower detection limit of the instrument. A beam split other than 50:50 can lead to problems if one ever needs to reference beam attenuate. Turbid samples can cause extreme problems in beam balancing in dual beam instruments.

        Dual Beam Instruments
        Bausch and Lomb
        Shimadzu 160

Double Beam:
The entire sample is diverted thru the blank and then the sample by means of the chopper. The optical bench may or may not have more optics then a single beam instrument, but have an additional motor needed to drive the chopper. The over all cost of the optical bench is greater but improved drift and S/N are obtained. One is not limited on the types of samples that can be run.

        Double Beam Instruments
        Perkin-Elmer
        Varian
        Shimadzu (265)


Photomultiplier Tubes vs Photodiodes and Phototubes:
Some manufactures use a photodiode as a detector in their optical system. This is not to be confused with a diode array. The optical bench has the normal configuration, either single beam or double beam, but they use a photodiode as the detector. The advantage again is reduced cost and a slightly longer wavelength range to about 1100 nm. They have less noise a high light levels so they show a slight advantage over PMT systems due to reduced noise at low absorbance. PMT's have a higher dark current and thus more inherent noise The disadvantage is their is no amplification of the signal by the detector as with a photomultiplier tube thus reduced S/N. PMT have far superior performance at low light levels, that is; high absorbance levels. The same is true of a phototube but they do not have the extended wavelength response.

关于杂散光的影响，下面也有精确的描述：

The Significance of Stray Light on Photometric Accuracy

The effect of stray light on photometric accuracy and the performance of a spectrophotometer can be quite dramatic. However with proper precautions, the photometric inaccuracy due to stray can be eliminated.  Let's consider the following points:

Definition - Stray light is any light outside the spectral region isolated by the monochromator that reaches the detector. It is produced by scatter from the optics and walls of the monochromator and is present in varying amounts in all spectrophotometers.

Stray light most frequently leads to deviations from the Beer-Lambert law and subsequent inaccuracy in photometric values. Double monochromator systems have lower stray light than a single monochromator but are as one would expect more expensive.

However, a number of questions arises:
    1. At what level does this increase in stray light become
       significant for the work one is doing?
    2. What level of error is associated with increasing stray?
    3. How much is one willing to pay for the difference?
    4. What are the trade offs between a single monochromator,
       double grating monochromator, and a double monochromator
       system?
    5. Are other option available to decrease stray light?

Let us consider the first question.

            A =  -log T
            Aobs = log [(1-T) S + T]      where S= stray light











Absorbance Observed

Level of Stray

     DM            DGM(Lambda 6HP)    SM(Lambda)           SM(DU)

Abs    .0001     %E        .0005     %E        0.02     %E        0.05    %E

1.0    1.000    .004        1.000    0.002        0.999    0.078        0.998    0.194
2.0    2.000    .002        1.997    0.017        1.991    0.425        1.979    1.048
3.0    2.999    .010        2.997    0.072        2.920    2.636        5.824    5.862
4.0    3.995    .110        3.978    0.529        3.522    11.92        3.222    19.44
5.0    4.958    .827        4.823    3.521        3.677    26.44        3.292    34.14

Where DM=Double Monochromator, DGM=Double Grating Monochromator, SM=Single Monochromator

As can be seen by Table 1 there is a difference, but the difference between S = 0.0001 and S = 0.0005 and S=0.05. However this difference is not significant until the absorbance exceeds 3 A.  It is of importance to compare the S=.0001 and S=0.001 levels. These two levels of stray light typically represent the levels between double monochromator systems (S = 0.0001) and a single monochromator system with a pre-grating system (S = 0.0005).  Stray light in standard configuration spectrophotometers (S = 0.02 or 0.05) does become high enough to prohibit their use at high absorbance.

It is also apparent that as the absorbance increases, the % error increases. Would one not be better off with a double monochromator system if they were measuring absorbances above 3A.  This is true.  However, one might want to consider the following table.

TABLE 2:

    Abs            %T
    O            1OO
    1            10
    2            1
    3            0.1
    4            0.01



At absorbance values above 3A one is limiting the performance of any spectrophotometer.  Since absorbance is a mathematical function of the amount of light passing through the sample (A =  - log T), what is one asking the instrument to read.  At 3A only 0.1% of the transmitted light is monitored and at 4A only 0.01%. One is requiring the instrument to accurately detect and compare values form the reference and sample beam that are at opposite extremes of its linear range. Cahill (1980) showed that this leads to increased noise in the spectrophotometer measurement.  This in itself will decrease the accuracy of the measurement by increasing the uncertainty due to noise.  Although one could use a reference beam attenuator to reduce noise, the preferred method is to simply dilute sample to a reasonable absorbance value for quantitative analysis or use short pathlength cells.

Another consideration that must be made between a single and a double monochromator instrument is the energy loss due to a double monochromator.  A conventional single monochromator's energy thru-put is typically about 50%.  If we place a pre-grating in the system it reduces the energy thru-put to about 40%.  A double monochromator system drops to about 22% thru-put. To compensate for this reduction in transmitted light, double monochromator systems usually resort to larger optics to increase the collection efficiency. The end result is that double monochromator system cost significantly more than single monochromator systems or double grating systems.

It would seem obvious that a trade-off is involved in any photometric system. For most applications, a single  monochromator system is all that is required to obtain accurate measurement. Single monochromator systems are limited to absorbance levels below 3A. Where high absorbing samples are encountered and sample dilutions is not possible, an instrument with higher stray light rejection is required. A double grating monochromator yields a significant reduction in stray. Double grating systems are lower in cost than a double monochromator system. Finally, some applications due require the use of a double monochromator system where ultimate accuracy is required.  A double monochromator offers the best stray light rejection but sacrifices energy thru put. This may limits the lower detection range of the instrument. However, remember it is not only the number but also the efficiency and more importantly the size of the optics that determine energy thru put. Each individual researcher will have to determine their needs and where necessary pay the additional expense of a double monochromator system.

1.  R. Mavrodineanu (1972) Accuracy in Spectorphotometric and Luminescence Measurements.  NBS Special Publication 378, U. S. Department of Commerce, National Bureau of Standards.
2.  J. Cahill  (198O) Noise in Double-Beam UV-VIS Spectrophotometers.

非常感谢auto1235,资料非常不错，收藏了。

原文由 tutm 发表:
没关系，如果结贴后没有理想的建议，我再往那儿发。现在别让这儿停顿下来。而且，看这一期的应该搞仪器的较多，了解这方面的版友更多些，我想机会也会多些。

这个问题，以前帖子里讨论过，spain那会也说过。

不过在网上找了下，还真没有同时具有测吸光度和荧光的仪器.

一个荧光仪如下图

我想，因为荧光仪的光源更容易激发产生荧光，那是不是可以通过在光源和第一个单色器之间加入滤光片，减少光强，减少产生荧光的可能。其他光路不改变，然后在样品室中，由于荧光是检测器成直角，所以吸光度时，在透射光的位置，放一个反光镜，反射透射光成直角进入检测器，如上图。

图中的polarize不知道什么作用，做成个可拆卸部件，测吸光度时拿走。

把荧光光谱仪改为分光光度计，可能会更容易些。

原文由 nemoium 发表:
这个问题，以前帖子里讨论过，spain那会也说过。

不过在网上找了下，还真没有同时具有测吸光度和荧光的仪器.

一个荧光仪如下图

我想，因为荧光仪的光源更容易激发产生荧光，那是不是可以通过在光源和第一个单色器之间加入滤光片，减少光强，减少产生荧光的可能。其他光路不改变，然后在样品室中，由于荧光是检测器成直角，所以吸光度时，在透射光的位置，放一个反光镜，反射透射光成直角进入检测器，如上图。

图中的polarize不知道什么作用，做成个可拆卸部件，测吸光度时拿走。

把荧光光谱仪改为分光光度计，可能会更容易些。

看来还不是简单的，比想象的复杂多了。
估计没戏。
本来我想如果有就多一个选择，买的时候可以兼顾一下，也许能省些钱。谢谢您为我的问题费心了

原文由 tutm 发表:
看来还不是简单的，比想象的复杂多了。
估计没戏。
本来我想如果有就多一个选择，买的时候可以兼顾一下，也许能省些钱。

呵呵，tutm老师的主人翁精神蛮强的。