CELL AND MOLECULAR BIOLOGY LABORATORY PRELIMINARY PERIOD EXPERIMENT 1 Use of Micropipettor and pectrop!oto"eter Micropipettor
1. The vol volume of air air spac space e in the barre rrel is ad adusted b! scre"in# the plun#er further in or out of the piston $. The The volum olume e is disp displa la! !ed on the di(ita& reado#t %. &epr &epres essi sin# n# the the plun# lun#er er "il "ill displace the specified volume of air from the piston '. eleas eleasin# in# the plun#er plun#er create creates s a )acc#"* $!ic! $i&& dra$ an e+#a e+#a&& )o&#" o&#"e e of f&#i f&#id d into t!e tip . *ithd *ithdra" ra"n n fluid fluid is e+pe e+pell lled ed b! depressin# the plun#er - If ther there e are are air air bubb bubblles, es, depr epress the plunger and and withdra withdraw w needed needed amount amount of volume (again) T,pes T, pes of Micropipettors 1- Red - ran# an#e is is 0.-1. -1.0 uL - tip 10 uL .- Ye&&o$ - ran# an#e is 10-100 100 uL - tip 100 uL /- B&#e - ran# ran#e e is 1000 1000-1 -100 000 0 uL - tip 1000 uL
Micropipettor is used to transfer volumes that are less than 1mL Measu easure res s as litt ittle as one microl microlite iterr (uL), (uL), one millio millionth nth (10^-6) of a litter Most Most usef useful ul unit units s of liqu liquid id meas measur urem ement ent in mole molecu cula lar r biology are milliliter (mL) and microliter (uL) Micropipettor is a precision p#"p $it! a disposa%&e tip 'o$ to #se t!e Micropipettor
UING T'E MICROPIPETTOR0
pectrop!oto"eter
1- i"p&e spectrop!oto"eter - /isible li#ht - %0 to 0 nm - Li#ht is produced b! a tun#sten lamp .- U2)is spectrop!oto"eter - 2econd lamp is used &34T354M turns a visible li#ht spectrophotometer into a 4/-visible unit that can measure from 170-%0 nm - 8vailable "ith a variet! of features scannin#, multiple cells, inter#ral printers, and user interfaces ABORBANCE AND CONCENTRATION O3 ABORBING MOLECULE 8bsorbance vs Transmittance A%sor%ance0 The amount of li#ht that can be absorbed Trans"ittance The amount of li#ht that passes throu#h the solution as it is not absorbed 8bsorbance rather than the transmittance is "ost #sef#& in spectrophotometr! If no &i(!t is a%sor%ed A%sor%ance 4 5 Trans"ittance 4 1556 3ach unit in absorbance corresponds "ith an order of ma#nitude in the fraction of li#ht transmitted 891, 10: of the li#ht is transmitted (0.10) 70: is absorbed 89$, 1: of the li#ht is transmitted and 77: is absorbed •
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Li#ht comes from a li#ht source (tun#sten lamp or delirium) then to a monochromator then lli#ht "ill pass throu#h the sample solution placed in a c#)ette and its "avelen#th "ill be in the di#ital displa! T,pes of c#)ette 1. lass cuvette $. lastiic cuvette
T,pes of pectrop!oto"eters
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89%, 0,1: of li#ht transmitted and 77,7: absorbed
is is
r4 Pearson9s corre&ation coefficient
3or"#&a0
Trans"ittance, T ! " ! # 6 Trans"ittance, $T %## T A%sor%ance,
& log %# ! # " ! & log %# % " T & log %# %## " $T & ' - log %# $T
7!at is t!e re&ation of Concentration and A%sor%ance8 3+plained b! the Beer9s La$ Beer9s La$ A4e%c *here 8 is absorbance (no units, since & log %# ! # " ! ) e is the molar absorbtivit! "ith units of L mol-1 cm-1 % is the path len#th of the sample - that is, the path len#th of the cuvette in "hich the sample is contained. *e "ill e+press this measurement
in
centimetres.
c is the concentration of the compound in solution, e+pressed in mol L-
8n increase in the absorbance "ill also lead to an increase in the concentration (direct relationship)
Linear Re(ression ,4": ; %
1- 7!, $as Bro"op!eno& %&#e =BPB> #sed in t!e e:peri"ent8 ;; "as used in the e+periment as an indicator
er or d!e .- 7!, $as )orte: #sed8 The vorte+ "as used to mi+ 8LL the components of the solution /- 7!at is t!e acc#rate a%sor%ance of Bro"op!eno& %&#e8 •
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ACCURACY PRECIION
Acc#rac, =o" close the measured value is to the actual value or true value Precision =o" close the value of the measurement is close to the other measured values b! the #roup
Ana&,tica& Ba&ance reat precision in ?uantitative chemical anal!sis C114C.. ;romophenol blue 1.$#BmL
