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Paper chromatography is an ancient technique to separate low molecular mass compounds based on their distribution between mobile phase (solvent) and stationary phase (cellulose and cellulose-bound water). Paper chromatography has been largely replaced by thin layer chromatography and high performance liquid chromatography as the latter methods have higher resolution capability. Nevertheless due to low cost and availability of great number of protocols for separation of various compounds, paper chromatography is still a powerful analytical tool. In the current protocol this technique is exemplified by separation of urocanic acid and deaminohistidine.

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Paper Chromatography as Exemplified by Separation of Urocanic Acid and Deaminohistidine
分离尿刊酸和多聚组氨酸的经典方法:纸色谱分析法

生物化学 > 其它化合物 > 生物碱
作者: Alexander V. Bogachev
Alexander V. BogachevAffiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia
For correspondence: bogachev@belozersky.msu.ru
Bio-protocol author page: a487
Vol 3, Iss 12, 6/20/2013, 3140 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.797

[Abstract] Paper chromatography is an ancient technique to separate low molecular mass compounds based on their distribution between mobile phase (solvent) and stationary phase (cellulose and cellulose-bound water). Paper chromatography has been largely replaced by thin layer chromatography and high performance liquid chromatography as the latter methods have higher resolution capability. Nevertheless due to low cost and availability of great number of protocols for separation of various compounds, paper chromatography is still a powerful analytical tool. In the current protocol this technique is exemplified by separation of urocanic acid and deaminohistidine.
Keywords: Paper chromatography(纸色谱), Urocanate(urocanate), Dihydrourocanate(dihydrourocanate)

[Abstract]

Materials and Reagents

  1. Chromatography paper (Whatman No. 1)
  2. Urocanic acid (MP Biomedicals)
  3. Deaminohistidine (Chem-Impex International)
  4. Isobutanol
  5. Acetone
  6. Formic acid
  7. Sulfanilic acid
  8. NaNO2
  9. HCl
  10. Na2CO3
  11. Pauly diazo reagent (see Recipes)

Equipment

  1. Chromatography chamber
  2. Capillary tubes (Kimble Chase Kimble, catalog number: 71900 10 )
  3. Spraying nozzle

Procedure

  1. Preconditioning of chromatography chamber
    1. Prepare chromatography solvent by mixing isobutanol, acetone, formic acid, and water in 160:160:1:39 proportion (vol. by vol.). 
    2. Pour the solvent into a chromatography chamber, the liquid level should be around 5 mm from the bottom. Seal the solvent-containing chamber for at least 1 h to saturate the chamber with solvent vapors.

  2. Preparation of chromatography paper
    1. Scissor out a strip of chromatography paper with the appropriate dimensions. The width of the strip depends on number of samples to be analyzed (the distance between sample spots and between spots and paper edges should be at least 20 mm). The height depends on difference in Rf values of separated compounds and usually is 100-400 mm.
    2. Draw a line from 20 mm from the bottom of the strip by a graphite pencil (starting line). Mark dots on this line for future sample spots.
    3. Load spots of the test samples and standards on the starting line using a capillary tube. Micro liter quantities are used to prepare spots. The diameter of the spots should be only up to a few mm. In order to get a concentrated spot, the spotting can be repeated several times. If it is necessary, wait till the spot is dried and then repeat the spotting. In the case of urocanic acid and deaminohistidine, each spot should contain about 10 nmol of one of these compounds.
       
  3. Chromatography
    1. To develop the chromatogram, place the prepared strip into the saturated chromatography chamber and seal the chamber.
    2. Remove the strip from the chamber when solvent front has traveled up to about 20 mm from the top of the paper. Mark position of the solvent front on the strip. 

  4. Detection
    1. Air dry the strip. Using a spraying nozzle spray the dried strip first with the Pauly diazo reagent until the paper become humid, followed by spraying the humid strip with a 10% Na2CO3 solution until spots of imidazolyl derivatives become colored (urocanic acid – orange, deaminohistidine – red). The color is stable within several weeks. The schematic representation of the typical chromatogram can be seen in Figure 1.
    2. Calculate Rf values [(distance traveled by a component)/(distance traveled by the solvent)].


      Figure 1. Schematic representation of the separation of urocanate and deaminohistidine by paper chromatography.


Recipes

  1. Pauly diazo reagent 
    Reagent A: Dissolve 0.9 g sulfanilic acid in H2O and add 9 ml concentrated HCl (37%), and then make up to a total volume of 100 ml in H2O
    Reagent B: Dissolve 5 g NaNO2 in 100 ml H2O
    Pauly diazo reagent: Mix 6 ml of ice cold reagent A with 6 ml of ice cold reagent B and incubate 5 min on ice. Add to the mixture additional 24 ml of reagent B and 64 ml of ice cold water. The Pauly diazo reagent can be stored for a few hours on ice.

Acknowledgments

This protocol was adapted from Sen et al. (1962). This work was supported by the Russian Foundation for Basic Research (project number 10-04-00352).

References

  1. Bogachev, A. V., Bertsova, Y. V., Bloch, D. A. and Verkhovsky, M. I. (2012). Urocanate reductase: identification of a novel anaerobic respiratory pathway in Shewanella oneidensis MR-1. Mol Microbiol 86(6): 1452-1463.
  2. Consden, R., Gordon, A. H. and Martin, A. J. (1944). Qualitative analysis of proteins: a partition chromatographic method using paper. Biochem J 38(3): 224-232.
  3. Sen, N. P., Mc, G. P. and Paul, R. M. (1962). Imidazolepropionic acid as a urinary metabolite of L-histidine. Biochem Biophys Res Commun 9: 257-261.

材料和试剂

  1. 色谱纸(Whatman 1号)
  2. 尿刊酸(MP Biomedicals)
  3. 脱氨基组氨酸(Chem-Impex International)
  4. 异丁醇
  5. 丙酮
  6. 甲酸
  7. 对氨基苯磺酸
  8. NaNO 2
  9. HCl
  10. Na 2 3
  11. Pauly重氮试剂(见配方)

设备

  1. 色谱室
  2. 毛细管(Kimble Chase Kimble,目录号:7190010)
  3. 喷嘴

程序

  1. 色谱室的预处理
    1. 通过混合160:160:1:39比例(体积比)的异丁醇,丙酮,甲酸和水来制备色谱溶剂。
    2. 将溶剂倒入色谱室,液面应距离底部约5 mm。 密封含溶剂的室至少1小时,使溶剂蒸汽饱和室
  2. 色谱纸的制备
    1. 剪下一条适当尺寸的色谱纸。 带的宽度取决于待分析的样品数量(样品点之间和点与纸张边缘之间的距离应为至少20mm)。 高度取决于分离的化合物的Rf值的差异,通常为100-400mm。
    2. 通过石墨铅笔(起始线)从带的底部20mm绘制线。在此行上标记点以便将来的样品点。
    3. 使用毛细管在起始线上加载测试样品和标准物的斑点。微升量用于制备斑点。斑点的直径应该只有几毫米。为了获得集中的斑点,斑点可以重复几次。如果有必要,等待现场干燥,然后重复点滴。在尿刊酸和脱氨基组氨酸的情况下,每个斑点应含有约10nmol的这些化合物之一。
       
  3. 色谱法
    1. 要开发色谱图,将准备的条放入饱和色谱室中并密封室。
    2. 当溶剂前沿从纸张顶部移动到约20 mm时,从样品室中取出样品条。标记条上溶剂前沿的位置。

  4. 检测
    1. 空气干燥钢带。 使用喷嘴,首先用Pauly重氮试剂喷雾干燥的条,直到纸变湿,然后用10%Na 2 CO 3溶液喷雾湿条,直到 咪唑基衍生物的斑点变成有色的(尿刊酸 - 橙色,脱氨基组氨酸 - 红色)。 颜色在几个星期内是稳定的。 典型色谱图的示意图如图1所示。
    2. 计算Rf值[(组分行进的距离)/(溶剂行进的距离)]。


      图1.通过纸色谱法分离尿刊酸和去氨基组氨酸的示意图


食谱

  1. Pauly重氮试剂
    试剂A:将0.9g对氨基苯磺酸溶解在H 2 O中并加入9ml浓HCl(37%),然后补充H 2 O中的总体积为100ml > O
    试剂B:将5g NaNO 2溶解在100ml H 2 O中。
    Pauly重氮试剂:将6ml冰冷的试剂A与6ml冰冷的试剂B混合,并在冰上孵育5分钟。 再向混合物中加入24ml试剂B和64ml冰冷的水。 Pauly重氮试剂可在冰上储存数小时。

致谢

该协议改编自Sen等人(1962)。这项工作是由俄罗斯基础研究基金会(项目号10-04-00352)支持。

参考文献

  1. Bogachev,A.V.,Bertsova,Y.V.,Bloch,D.A。和Verkhovsky,M.I。(2012)。 尿酸还原酶:在希瓦氏菌中鉴定新的无氧呼吸途径 MR -1。 Mol Microbiol 86(6):1452-1463。
  2. Consden,R.,Gordon,A.H.and Martin,A.J。(1944)。 蛋白质的定性分析:使用纸张的分区色谱法。 Biochem J 38(3):224-232。
  3. Sen,N.P.,Mc,G.P.and Paul,R.M。(1962)。 咪唑丙酸作为L-组氨酸的尿代谢物。 Biochem Biophys Res Commun 9:257-261。
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How to cite this protocol: Bogachev, A. V. (2013). Paper Chromatography as Exemplified by Separation of Urocanic Acid and Deaminohistidine. Bio-protocol 3(12): e797. DOI: 10.21769/BioProtoc.797; Full Text



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