Question

if the temperature of the column in HPLC was to increase by <1 degree celcius, what effect would this have on the data?

why do the mean and standard deviation differ between different concentrations when using HPLC?

why does assay range for a drug differ in the British Pharmacopoeia and the Ph Eur?

Answer 1

Solution:

1. Increase in column temperature will result in increate in rate chromatographic separation. In general, for a reverse-phase isocratic chromatographic separation a retention time decrease of 1–2% for every 1°C column temperature increase. A small variation of the column temperature will affect several factors such as retention time of sample, compounds those have close retention time may elute at same retention time by even a small cahnge in the column temperature.  

2.

Mean: x̄

x̄= (X1 + X2 + ···Xn) / n

A sample of same concentration is to be injected n times and its area for each injection has to be measured and by above formula.

X1 = Area of injection 1 of sample having same concentration.

X2 = Area of injection 2 of sample having same concentration.

If the samples having different concentrations then obviosly means for each concentration samples will be different as area changes as concentration changes.

Standard Deviation [s]

Formula for standard deviation; s = [ ∑ (xi-x̄)2 / (n-1) ]1/2

xi = Area of ith injection of sample having same concentration

x̄ = mean of areas of injection of sample having same concentration

n = number of injections of sample having same concentration.

If the samples having different concentrations then obvioisly standard deviation for each concentration samples will be different as area changes as concentration changes.

Mean and standard deviation is explained by following representative example for a sample having same concentration;

injections Area

1 1110

2 1113

3 1111

4 1114

Mean

x̄ = (1110 + 1117 + 1109 + 1114) / 4

x̄ = 1112.5

Standard deviataion for all injection

s = [ ∑ (xi-x̄)2 / (n-1) ]1/2

s = {[ (1110-1112.5)2 + (1117 -1112.5)2 + (1109-1112.5)2 + (1114- 1112.5)2 / (4-1) }1/2

s = {[(-2.5)2 + (4.5)2 + (-3.5)2 +(1.5)2] / (4-1) }1/2

s = {[(-6.25) + (20.25) + (-12.25) +(2.25)] / (4-1) }1/2

s = [4/3]1/2

s = 1.15

3. According to our knowledge there is differ in assay range of drug in British pharmacopoeia and Europe pharmacopoeia. Since, there is no monograph for drugs in Europe pharmacopoeia, for assay range of drug Europe pharmacopoeia is also following guidlines of British pharmacopoeia. If there is difference for assay of drug between British pharmacopoeia and Europe pharmacopoeia. Then it will beacuse of they kept their own limits for the assay of drug.

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