Photodocu mentation Procedu res

4. Photodocu mentation Procedu res

1 ) Meter l i ghting to determ ine exposure

The expos u re is determ ined by TTL (through the lens) meteri ng, using the built-in light meter of the camera. The reflected light from the TLC plate can alternatively be metered with a handheld light mete r. I t is reco m ­ mended that t h e f-stop b e set a t f 8.0-which provides a good depth

of field-and that the expos u re time be adjusted until the proper expos u re

i s reached .

2) Photograph and bracket shots Make sure that the camera ASA is set to the appropriate ASA for the fi lm being used and that the f-stop is set at f 8.0. Photograph the plate with the exposure time set to the metered read i ng. Bracket the ex posu re by changing the f-stop u p one stop, then down one stop, from the metered exposure .

Photodocumentation of the TLC Plate Using Visible Light 129

exception to this rule is the photodocumentation of proteins detected by

ninhydrin. In this case, the separation zones are more easily seen a fter 24 hours. TL C plates for protein analysis should be photographed between 24

and 48 hours after detection.)

3) Document and l og each shot It is advisable to keep a writte n document of the photographs. This i nfor­ mation can be kept in a laboratory notebook, in a ri ng binder, or on a com puter database. The information is val u able i n that it provides documen­

tation on the appropriate ex posu re for the photograph. If the photo -

graphs do not come out satisfactori ly, the i nformation can be useful in making expos u re corrections. It also al lows for tracking of photographs, par­ ticularly for multiple origi nals that may be simi lar. Most i m portant, should the photograph need to be dupl icated at a later date, all necessary infor­ m ation on the exposure has been retai ned. A sample log s heet is attached (Attachment A) .

4) Rewind film and send for development After all expos u res are made, rewind and remove the film from the camera. Send the film to a reliable photo processi n g laboratory. Ask the lab to n u m ber the slides, as this helps correlate each s hot with written documen­

tatio n . Black-and - wh ite fi l m is processed and pri nted as a proof sheet.

Individual photographs can be chosen for printing from the proof sheet or the slide.

5) Label and store sl ides and pri nts Labeling of slides is strongly enco u raged. This is particularly useful when slides are borrowed by colleagues fo r presentations, so that the slides can be easi ly refi led . Proof s heets should be placed in 8 x 1 0" polyethylene sheet

protectors , and negatives can be housed in negative hold ers . The proof sh eets and the negatives can then be stored in D-ring binders . Slide labels can be handwritte n , typed , or generated by com puter. The label should contai n at least: Name, Date, and Subiect. Other useful i nfor­ mation can be added to the label, as wel l . A sample label may look like this:

Photo No. Subject Camera

Date

Roli No.

Shutter speed Film

PRO Laser labels are designed to fit slide mou nts and can be pri nted on laser pri nters . They can be obtai ned through ph otographic su ppliers or o rdered

d i rectly from the manufactu rer. For direct orders, write to: Slide Scri be, 752 Wash i ngton Avenue So., Min neapolis, MN 55439.

Helpful Hint: A dot can be placed in the upper right corner of each label and

Striegel . H i l l

slide. When a l l slides are properly placed i n the slide carousel, the dot will

be seen on the outer edge of the slide. Always use polyethylene holders for photographic i mages , as nonarchival

materials will damage the image. For val uable slides, make d u plicates and store one copy in a separate place.

Attachment A Photodocumentation Log Sheet

Film rol l no.

Su bject

Correction filters

Polaroid

Diagram of eq uipment setup

Photodocumentation of the TLe Plate Using Visible Light

131 Name

Film roll no. #

Sample Camera distance

f-stop

Sh utter

Filter

Description Comments

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

Protocol G

Photodocumentation of the TLC Plate Using Ultraviolet Light

Summary Guideli nes for photographic documentation of a TLC plate by ultraviolet fluorescence photography

Equipment:

35-mm camera Copy stand Ultraviolet light box (see Attachment A)

Two handheld u ltraviolet lamps Light meter (optional)

Filters:

Kodak Wratten gel filters (2 E , 1 0R, 1 0M , 20Y)

Film:

Black-and-white film: 3 5 - m m Kodak TMAX 400 Color slide film: 3 5 - m m Fujichrome Professional 400

Time: Approximately 30 minutes per plate (less i n a large series)

Scope Photography is u sed to q u ickly and accu rately record the TLC resu lts under either visible or ultraviolet light. This protocol describes the proced u res used to

photograph a TLC plate under u ltraviolet light. For photod ocu me ntation of TLC plates u nder visi ble light, see Protocol F.

Photography is the preferred method for permanently record ing the resu lts of

a TLC experi ment. TLC plates detected with u ltraviolet fluorescent reagents, such as those u sed for the analysis of carbo hyd rates, waxes , and resins, are photo­ graphed u nder the i l l u m i nation of u ltraviolet lamps. The separation zones of the chromatogram are seen as visi ble fl uorescent spots. The TLC plate is photo­ graphed with black-and-wh ite print film and color slide film u nder ultraviolet i l l u ­

m i nation , u s i n g a 3 5 - m m camera on a copy stand .

Striegel . Hill

Scheme Documentation of Thin-Layer Chromatography by

U ltraviolet Fluorescence Photography

Supporting Operation

Main Operation