please help me

[mazan2010]

hello
i'm mazan from Egypt , i need your help
i working on quality control
we do determination of molecular size distribution of polysaccharide antigen by using column chromatography.
after this we take this fraction from the column then measure every fraction with spectrophotometer ,
then we plot volume against absorbance , to get the high peak then measure
area under curve for this
we make this manually ,i want make this with any software to get accurate result
if any one can help me how i can do this
contact with me on my mail
wtwm2003@yahoo.com
thanks

[Alfred88]

Dear mazan2010:
There are some software packages to help you with the SEC calculations. You have to purchase a set of standards (e.g. Polystyrene calibration stds). Basically, you need to have Mw and Mn values for each standard.

Please check out the Clarity software (free trial download). It has a module for SEC calculations. I read that Chromeleon 6.8 also has this function, but never use it.

[Noser222]

If you're doing all this manually you don't need any software other than a spreadsheet to plot your data.

Calibrate with standards as Alfred88 said. In your case, for a polysaccharide, pullulan standards may work. Plot the log MW vs. elution volume.

[mazan2010]

thanks Alfred88 and Noser222 to answer me
i will explain to you what i do with some data for example to learn me how i can do this with any software you can tell and send me link for it.
we make quality control for product as followed:
we take the product and dissolve it in injection buffer (1ml) which consist of elution buffer and contain sodium azide then apply this to column chromatography then recive the eluted in test tube then measure this with spectrophotometer , from this data we determine Kd for the polysacchride in product as the following equation
kd= (Ve-Vo)/(Vt-Vo)
Ve is elution volume for the polysacchirde
Vo is elution volume for polydextran (calibration standard)
Vt is elution volume for sodium azide
this Kd must be equal to 0,4 or less
then we plot optical density on Y axis against volume on X axis to calculate
recovery percentage assuming that Kd equal or smaller than 0,5 at this equation
(area in evaluated peak of polysacchride at Kd equal 0,5/total area in evaluated peak)
and the result multiply it with 100 to get percentage
this all work

2 = 0.447
4= 0.391
6 =0.38
8= 0.327
10= 0.387
12 =0.366
14 =0.376
16 =0.33
18 =0.385
20 =0.361
22 =0.378
24 =0.347
26= 0.527
28 =0.672
30 =0.732
32= 0.436
34 =0.448
36= 0.399
38 =0.465
40 =0.428
42 =0.444
44 =0.428
46 =0.461
48 =0.451
50 =0.453
52 =0.443
54 =0.464
56 =0.422
58 =0.449
60 =0.405
62 =0.426
64 =0.373
66 =0.396
68 =0.358
70 =0.387
72 =0.329
74 =0.364
76 =0.333
78 =0.372
80 =0.338
82 =0.39
84 =0.356
86 =0.371
88 =0.341
90 =0.395
92 =0.471
94 =0.438
96 =0.519
98 =0.469
100 =0.573
102 =0.596
104 =0.408
106 =0.475
108 =0.36
110 =0.397
112 =0.342
114 =0.377
116 =0.335
118 =0.411
120 =0.344
please this is the data help me to how i can calculate with any software and send this link for this software

[Alfred88]

Dear mazan2010:
If your procedure is "home-brew" SOP, you need to talk to the author/developer to get insights/training.
If your procedure is 'official' in BP/EP/USP/JP, you can either:
1/contact a contract lab to outsource this task
2/post more info on this 'official' method, on this forum.

BTW, two more suggestions:
1/It is hard to understand your written description well. Please ask somebody to edit your writing.
2/Use a catchy title, e.g. 'problem with calc. for Polydextrans'

[Don_Hilton]

Plotting the data, I can see the sodium Azide peak clearly, but the baseline is noisy and the polysaccharide and polydextran peaks are a problem. (In looking at the plot -- umm... I think I had the order backwards... note added to post 4/21)



It looks like you need to look at the procedure you use for collecting fractions and reading absorbance -- to reduce the noise. With a better plot (either more data points to allow smoothing or less noise) it may be easier to see the retention volumes of all the compunds.